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The anterior pituitary gland drives highly conserved physiologic processes in mammalian species. These hormonally controlled processes are central to somatic growth, pubertal transformation, fertility, lactation, and metabolism. Current cellular models of mammalian anteiror pituitary, largely built on candidate gene based immuno-histochemical and mRNA analyses, suggest that each of the seven hormones synthesized by the pituitary is produced by a specific and exclusive cell lineage. However, emerging evidence suggests more complex relationship between hormone specificity and cell plasticity. Here we have applied massively parallel single-cell RNA sequencing (scRNA-seq), in conjunction with complementary imaging-based single-cell analyses of mRNAs and proteins, to systematically map both cell-type diversity and functional state heterogeneity in adult male and female mouse pituitaries at single-cell resolution and in the context of major physiologic demands. These quantitative single-cell analyses reveal sex-specific cell-type composition under normal pituitary homeostasis, identify an array of cells associated with complex complements of hormone-enrichment, and undercover non-hormone producing interstitial and supporting cell-types. Interestingly, we also identified a Pou1f1-expressing cell population that is characterized by a unique multi-hormone gene expression profile. In response to two well-defined physiologic stresses, dynamic shifts in cellular diversity and transcriptome profiles were observed for major hormone producing and the putative multi-hormone cells. These studies reveal unanticipated cellular complexity and plasticity in adult pituitary, and provide a rich resource for further validating and expanding our molecular understanding of pituitary gene expression programs and hormone production. Electronic supplementary material The online version of this article (10.1007/s13238-020-00705-x) contains supplementary material, which is available to authorized users.

We previously demonstrated that the two paralogous RNA-binding proteins PCBP1 and PCBP2 are individually essential for mouse development: Pcbp1-null embryos are peri-implantation lethal, while Pcbp2-null embryos lose viability at midgestation. Midgestation Pcbp2(−/−) embryos revealed a complex phenotype that included loss of certain hematopoietic determinants. Whether PCBP2 directly contributes to erythropoietic differentiation and whether PCBP1 has a role in this process remained undetermined. Here, we selectively inactivated the genes encoding these two RNA-binding proteins during differentiation of the erythroid lineage in the developing mouse embryo. Individual inactivation of either locus failed to impact viability or blood formation. However, combined inactivation of the two loci resulted in midgestational repression of erythroid/hematopoietic gene expression, loss of blood formation, and fetal demise. Orthogonal ex vivo analyses of primary erythroid progenitors selectively depleted of these two RNA-binding proteins revealed that they mediate a combination of overlapping and isoform-specific impacts on hematopoietic lineage transcriptome, impacting both mRNA representation and exon splicing. These data lead us to conclude that PCBP1 and PCBP2 mediate functions critical to differentiation of the erythroid lineage.

The establishment of efficient and stable splicing patterns in terminally differentiated cells is critical to maintenance of specific functions throughout the lifespan of an organism. The human α-globin (hα-globin) gene contains 3 exons separated by 2 short introns. Naturally occurring α-thalassemia mutations that trigger aberrant splicing have revealed the presence of cryptic splice sites within the hα-globin gene transcript. How cognate (functional) splice sites are selectively used in lieu of these cryptic sites has remained unexplored. Here we demonstrate that the preferential selection of a cognate splice donor essential to functional splicing of the hα-globin transcript is dependent on the actions of an intronic cytosine (C)-rich splice regulatory determinant and its interacting polyC-binding proteins. Inactivation of this determinant by mutation of the C-rich element or by depletion of polyC-binding proteins triggers a dramatic shift in splice donor activity to an upstream, out-of-frame, cryptic donor. The essential role of the C-rich element in hα-globin gene expression is supported by its coevolution with the cryptic donor site in primate species. These data lead us to conclude that an intronic C-rich determinant enforces functional splicing of the hα-globin transcript, thus acting as an obligate determinant of hα-globin gene expression.

BackgroundThe pituitary gland is a neuroendocrine organ containing diverse cell types specialized in secreting hormones that regulate physiology. Pituitary thyrotropes produce thyroid-stimulating hormone (TSH), a critical factor for growth and maintenance of metabolism. The transcription factors POU1F1 and GATA2 have been implicated in thyrotrope fate, but the transcriptomic and epigenomic landscapes of these neuroendocrine cells have not been characterized. The goal of this work was to discover transcriptional regulatory elements that drive thyrotrope fate.ResultsWe identified the transcription factors and epigenomic changes in chromatin that are associated with differentiation of POU1F1-expressing progenitors into thyrotropes using cell lines that represent an undifferentiatedPou1f1lineage progenitor (GHF-T1) and a committed thyrotrope line that produces TSH (TαT1). We compared RNA-seq, ATAC-seq, histone modification (H3K27Ac, H3K4Me1, and H3K27Me3), and POU1F1 binding in these cell lines. POU1F1 binding sites are commonly associated with bZIP transcription factor consensus binding sites in GHF-T1 cells and Helix-Turn-Helix (HTH) or basic Helix-Loop-Helix (bHLH) factors in TαT1 cells, suggesting that these classes of transcription factors may recruit or cooperate with POU1F1 binding at unique sites. We validated enhancer function of novel elements we mapped nearCga, Pitx1, Gata2,andTshbby transfection in TαT1 cells. Finally, we confirmed that an enhancer element nearTshbcan drive expression in thyrotropes of transgenic mice, and we demonstrate that GATA2 enhancesTshbexpression through this element.ConclusionThese results extend the ENCODE multi-omic profiling approach to the pituitary gland, which should be valuable for understanding pituitary development and disease pathogenesis.Graphical abstract

Pituitary thyrotropes are specialized cells that produce thyroid stimulating hormone (TSH), a critical factor for growth and maintenance of metabolism. The transcription factors POU1F1 and GATA2 have been implicated in thyrotrope fate and transcriptional regulation of the beta subunit of TSH,Tshb, but no transcriptomic or epigenomic analyses of these cells has been undertaken. The goal of this work was to discover key transcriptional regulatory elements that drive thyrotrope fate. We identified the transcription factors and epigenomic changes in chromatin that are associated with differentiation of POU1F1-expressing progenitors into thyrotropes, a process modeled by two cell lines: one that represents an early, undifferentiatedPou1f1lineage progenitor (GHF-T1) and one that is a committed thyrotrope that produces TSH (TαT1). We generated and compared RNA-seq, ATAC-seq, histone modification (including H3K27Ac, H3K4Me1, and H3K27Me3), and transcription factor (POU1F1) binding in these two cell lines to identify regulatory elements and candidate transcriptional regulators. We identified POU1F1 binding sites that were unique to each cell line. POU1F1 binding sites are commonly associated with bZIP transcription factor consensus binding sites in GHF-T1 cells and Helix-Turn-Helix (HTH) or basic Helix-Loop-Helix (bHLH) factors in TαT1 cells, suggesting that these classes of transcription factors may recruit or cooperate with POU1F1 binding to unique sites. We validated enhancer function of novel elements we mapped nearCga, Pitx1, Gata2,andTshbby transfection in TαT1 cells. Finally, we confirmed that an enhancer element nearTshbcan drive expression in thyrotropes of transgenic mice, and we demonstrate that GATA2 enhancesTshbexpression through this element. These results extend the ENCODE multi-omic profiling approach to an organ that is critical for growth and metabolism, which should be valuable for understanding pituitary development and disease pathogenesis.

A CRISPR screen for RBC regulators Hemoglobin in red blood cells (RBCs) carries oxygen to the tissues. Sickle cell disease is an inherited condition that involves abnormal hemoglobin. Current treatments entail modulating the level of fetal hemoglobin expression. Grevet et al. performed a CRISPR-Cas9 screen for regulators of fetal hemoglobin in RBCs and identified heme-regulated eIF2α kinase (HRI). Depleting the kinase in RBCs led to an increase in fetal hemoglobin levels and reduced sickling of cultured human RBCs. Thus, HRI may be a therapeutic target for sickle cell disease and other hemoglobin disorders. Science , this issue p. 285

The PolyC binding proteins (PCBPs) impact alternative splicing of a subset of mammalian genes that are enriched in basic cellular functions. Here, we focus our analysis on PCBP-controlled cassette exon-splicing within the cell cycle control regulator cyclin-dependent kinase-2 (CDK2) transcript. We demonstrate that PCBP binding to a C-rich polypyrimidine tract (PPT) preceding exon 5 of the CDK2 transcript enhances cassette exon inclusion. This splice enhancement is U2AF65-independent and predominantly reflects actions of the PCBP1 isoform. Remarkably, PCBPs’ control of CDK2 ex5 splicing has evolved subsequent to mammalian divergence via conversion of constitutive exon 5 inclusion in the mouse CDK2 transcript to PCBP-responsive exon 5 alternative splicing in humans. Importantly, exclusion of exon 5 from the hCDK2 transcript dramatically represses the expression of CDK2 protein with a corresponding perturbation in cell cycle kinetics. These data highlight a recently evolved post-transcriptional pathway in primate species with the potential to modulate cell cycle control.

Analysis of PIP-seq data from a model of erythropoiesis reveals dynamics of RBP–RNA interaction and changes in RNA secondary structure throughout red blood cell development., Two features of eukaryotic RNA molecules that regulate their post-transcriptional fates are RNA secondary structure and RNA-binding protein (RBP) interaction sites. However, a comprehensive global overview of the dynamic nature of these sequence features during erythropoiesis has never been obtained. Here, we use our ribonuclease-mediated structure and RBP-binding site mapping approach to reveal the global landscape of RNA secondary structure and RBP–RNA interaction sites and the dynamics of these features during this important developmental process. We identify dynamic patterns of RNA secondary structure and RBP binding throughout the process and determine a set of corresponding protein-bound sequence motifs along with their dynamic structural and RBP-binding contexts. Finally, using these dynamically bound sequences, we identify a number of RBPs that have known and putative key functions in post-transcriptional regulation during mammalian erythropoiesis. In total, this global analysis reveals new post-transcriptional regulators of mammalian blood cell development.

Differentiation of the hormone-producing cells of the pituitary represents an informative model of cell fate determination. The generation and maintenance of 2 pituitary lineages, the growth hormone (GH)- producing somatotropes and the prolactin (PRL)- producing lactotropes, are dependent on the pituitary-specific transcription factor, POU1F1. While POU1F1 is expressed in both cell types, and plays a role in activation of both the Gh and Prl genes, expression of Gh and Prl is restricted to somatotropes and lactotropes, respectively. These observations imply the existence of additional factors that contribute to the somatotrope and lactotrope identities and their hormone expressions. Prior transcriptome analysis of primary somatotropes and lactotropes isolated from the mouse pituitary identified enrichment of a transcription factor, Nr4a2, in the lactotropes. Nr4a2 was shown in a cell culture model to bind the Prl promoter at a position adjacent to Pou1f1 and to synergize with Pou1f1 in driving Prl transcription. Here we demonstrate in vivo the role of Nr4a2 as an enhancer of Prl expression by conditional gene inactivation of the Nr4a2 gene in mouse lactotropes. We demonstrate that nuclear orphan receptor transcription factor (NR4A2) binding at the Prl promoter is dependent on actions of POU1F1; while POU1F1 is essential to loading polymerase (Pol) II on the Prl promoter, Nr4a2 plays a role in enhancing Pol II release into the Prl gene body. These studies establish an in vivo role of Nr4a2 in enhancing Prl expression in mouse lactotropes, explore its mechanism of action, and establish a system for further study of the lactotrope lineage in the pituitary.

The anterior pituitary gland drives a set of highly conserved physiologic processes in mammalian species. These hormonally-controlled processes are central to somatic growth, pubertal transformation, fertility, lactation, and metabolism. Current models, largely built upon candidate gene based immuno-histochemical and mRNA analyses, suggest that each of the seven hormones synthesized by the pituitary is produced by a specific and exclusive cell lineage. However, emerging evidence suggests more complex models of hormone specificity and cell plasticity. Here we have applied massively parallel single-cell RNA sequencing (scRNA-seq), in conjunction with a set of orthogonal mRNA and protein imaging studies, to systematically map the cellular composition of adult male and female mouse pituitaries at single-cell resolution and in the setting of major physiologic demands. These analyses reveal sex-specific cellular diversity associated with normal pituitary homeostasis, and identify an array of cells with complex complements of hormone-enrichment as well as a series of non-hormone producing interstitial and supporting cell lineages. These scRNA-seq studies identify a major cell population that is characterized by a unique multi-hormone gene expression profile. The detection of dynamic shifts in cellular representations and transcriptome profiles in response to two well-defined physiologic stresses suggests corresponding roles of a number of these clusters in cellular plasticity within the adult pituitary. These studies point to an unanticipated complexity and plasticity in pituitary cellular composition that expands upon current models and concepts of pituitary gene expression and hormone production.

The differentiation of the hormone-producing cell lineages of the anterior pituitary represents an informative model of mammalian cell fate determination. The generation and maintenance of two of these lineages, the GH-producing somatotropes and prolactin (PRL)-producing lactotropes, are dependent on the pituitary-specific transcription factor POU1F1. Whereas POU1F1 is expressed in both cell types, and plays a direct role in the activation of both the Gh and Prl genes, GH expression is restricted to somatotropes and PRL expression is restricted to lactotropes. These observations imply the existence of additional, cell type-enriched factors that contribute to the somatotrope and lactotrope cell identities. In this study, we use transgenic mouse models to facilitate sorting of somatotrope and lactotrope populations based on the expression of fluorescent markers expressed under Gh and Prl gene transcriptional controls. The transcriptomic analyses reveal a concordance of gene expression profiles in the two populations. The limited number of divergent mRNAs between the two populations includes a set of transcription factors that may have roles in pituitary lineage divergence and/or in regulating expression of cell type-specific genes after differentiation. Four of these factors were validated for lineage enrichment at the level of protein expression, two somatotrope enriched and two lactotrope enriched. Three of these four factors were shown to have corresponding activities in appropriate enhancement or repression of landmark genes in a cell culture model system. These studies identify novel regulators of the somatotropes and lactotropes, and they establish a useful database for further study of these lineages in the anterior pituitary.

Formation of the mammalian hematopoietic system is under a complex set of developmental controls. Here, we report that mouse embryos lacking the KH domain poly(C) binding protein, Pcbp2, are selectively deficient in the definitive erythroid lineage. Compared to wild-type controls, transcript splicing analysis of the Pcbp2-/- embryonic liver reveals accentuated exclusion of an exon (exon 6) that encodes a highly conserved transcriptional control segment of the hematopoietic master regulator, Runx1. Embryos rendered homozygous for a Runx1 locus lacking this cassette exon (Runx1ΔE6) effectively phenocopy the loss of the definitive erythroid lineage in Pcbp2-/- embryos. These data support a model in which enhancement of Runx1 cassette exon 6 inclusion by Pcbp2 serves a critical role in development of hematopoietic progenitors and constitutes a critical step in the developmental pathway of the definitive erythropoietic lineage.

International audience; POU1F1, a pituitary-specific POU-homeo domain transcription factor, plays an essential role in the specification of the somatotroph, lactotroph and thyrotroph lineages and in the activation of GH1, PRL and TSHβ transcription. Individuals with mutations in POU1F1 present with combined deficiency of GH, PRL and TSH. Here, we identified a heterozygous missense mutation with evidence of pathogenicity, at the POU1F1 locus, in a large family in which an isolated growth hormone deficiency segregates as an autosomal dominant trait. The corresponding p.Pro76Leu mutation maps to a conserved site within the POU1F1 transactivation domain. Bandshift assays revealed that the mutation alters wild-type POU1F1 binding to cognate sites within the hGH-LCR and hGH1 promoter, but not to sites within the PRL promoter, and it selectively increases binding affinity to sites within the hGH-LCR. Co-immunoprecipitation studies reveal that this substitution enhances interactions of POU1F1 with three of its cofactors, PITX1, LHX3a and ELK1, and that residue 76 plays a critical role in these interactions. The insertion of the mutation at the mouse Pou1f1 locus results in a dramatic loss of protein expression despite normal mRNA concentrations. Mice heterozygous for the p.Pro76Leu mutation were phenotypically normal while homozygotes demonstrated a dwarf phenotype. Overall, this study unveils the involvement of POU1F1 in dominantly inherited isolated GH deficiency and demonstrates a significant impact of the Pro76Leu mutation on DNA-binding activities, alterations in transactivating functions and interactions with cofactors. Our data further highlight difficulties in modeling human genetic disorders in the mouse despite apparent conservation of gene expression pathways and physiologic functions.

The functions of the major mammalian cytoplasmic poly(A) binding protein, PABPC1, have been characterized predominantly in the context of its binding to the 3′ poly(A) tails of mRNAs. These interactions play important roles in post-transcriptional gene regulation by enhancing translation and mRNA stability. Here, we performed transcriptome-wide CLIP-seq analysis to identify additional PABPC1 binding sites within genomically encoded mRNA sequences that may impact on gene regulation. From this analysis, we found that PABPC1 binds directly to the canonical polyadenylation signal in thousands of mRNAs in the mouse transcriptome. PABPC1 binding also maps to translation initiation and termination sites bracketing open reading frames, exemplified most dramatically in replication-dependent histone mRNAs. Additionally, a more restricted subset of PABPC1 interaction sites comprised A-rich sequences within the 5′ UTRs of mRNAs, including Pabpc1 mRNA itself. Functional analyses revealed that these PABPC1 interactions in the 5′ UTR mediate both auto- and trans-regulatory translational control. In total, these findings reveal a repertoire of PABPC1 binding that is substantially broader than previously recognized with a corresponding potential to impact and coordinate post-transcriptional controls critical to a broad array of cellular functions.

Fundacao para a Ciencia e a Tecnologia (FCT) [POCI/BIA-BCM/59140/2004 and UID/MULTI/04046/2013 to BioISI from FCT/MCTES/PIDDAC; SFRH/BD/14273/2003 to F.J.C.P. and SFRH/BD/63581/2009 to C.B.]. Funding for open access charge: Instituto Nacional de Saude Doutor Ricardo Jorge and Oxford Journals Publisher. Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature termination codons (PTCs). The level of sensitivity of a PTC-containing mRNA to NMD is multifactorial. We have previously shown that human β-globin mRNAs carrying PTCs in close proximity to the translation initiation AUG codon escape NMD. This was called the 'AUG-proximity effect'. The present analysis of nonsense codons in the human α-globin mRNA illustrates that the determinants of the AUG-proximity effect are in fact quite complex, reflecting the ability of the ribosome to re-initiate translation 3′ to the PTC and the specific sequence and secondary structure of the translated ORF. These data support a model in which the time taken to translate the short ORF, impacted by distance, sequence, and structure, not only modulates translation re-initiation, but also impacts on the exact boundary of AUG-proximity protection from NMD. publishersversion published

The transcription factor Pit-1 (POU1-F1) plays a dominant role in cell lineage expansion and differentiation in the anterior pituitary. Prior studies of the mouse Pit-1 (mPit-1) gene revealed that this master regulatory locus is activated at embryonic day 13.5 (E13.5) by an early enhancer (EE), whereas its subsequent expression throughout adult life is maintained by a more distal definitive enhancer (DE). Here, we demonstrate that the sequential actions of these two enhancers are linked to corresponding shifts in their proximities to the Pit-1 promoter. We further demonstrate that the looping of the definitive enhancer to the mPit-1 promoter is critically dependent on a self-sustaining autoregulatory mechanism mediated by the Pit-1 protein. These Pit-1-dependent actions are accompanied by localized recruitment of CBP and enrichment for H3K27 acetylation within the Pit-1 locus. These data support a model in which the sequential actions of two developmentally activated enhancers are linked to a corresponding shift in higher-order chromatin structures. This shift establishes an autoregulatory circuit that maintains durable expression of Pit-1 throughout adult life.

The human growth hormone (hGH) gene is controlled by a long-range enhancer, HSI, located 14.5 kb 5′ to the hGH promoter. HSI establishes a domain of noncoding transcription that is ‘looped’ to the hGH promoter as an essential step in initiating hGH gene expression. Thus, defining how HSI generates its domain of noncoding transcription is central to understanding its long-range function. Here, we demonstrate that activation of noncoding transcription reflects an HSI-autonomous activity fully independent of interactions with linked gene promoters and occurring in spatial and temporal synchrony with initiation of GH expression in the embryonic pituitary. HSI establishes its noncoding transcription start sites (TSS) over a defined distance from its core determinants and in a manner independent of local primary sequences. The interval between HSI and it TSS co-maps with a domain of disordered and/or highly mobile nucleosomes specific to the pituitary locus. Thus, a localized chromatin reconfiguration by HSI and consequent establishment of an adjacent domain of noncoding transcription constitute initiating events in long-range enhancer function within the hGH locus.

The robust and tissue-specific activation of the human growth hormone (hGH) gene cluster in the pituitary and placenta constitutes an informative model for analysis of gene regulation. The five-gene hGH cluster is regulated by two partially overlapping sets of DNase I hypersensitive sites (HSs) that constitute the pituitary (HSI, II, III and V) and placental (HSIII, IV, and V) locus control regions (LCRs). The single placenta-specific LCR component, HSIV, is located at -30 kb to the cluster. Here we generate a series of hGH/BAC transgenes specifically modified to identify structural features of the hGH locus required for its appropriate placental expression. We find that placental specificity is dependent on the overall multigene configuration of the cluster whereas the distance between the cluster and its LCR impacts the level of placental expression. We further observe that a major function of the placental hGH LCR is to insulate the transgene locus from site-of-integration effects. This insulation activity is linked to placenta-specific occupancy of the chromatin architectural protein, CTCF, at HSIV. These data reveal a remarkable combination of structural configurations and regulatory determinants that must work in concert to insure robust and tightly controlled expression from a complex multigene locus.

We have previously demonstrated that the KH-domain protein αCP binds to a 3′ untranslated region (3′UTR) C-rich motif of the nascent human alpha-globin (hα-globin) transcript and enhances the efficiency of 3′ processing. Here we assess the genome-wide impact of αCP RNA-protein (RNP) complexes on 3′ processing with a specific focus on its role in alternative polyadenylation (APA) site utilization. The major isoforms of αCP were acutely depleted from a human hematopoietic cell line, and the impact on mRNA representation and poly(A) site utilization was determined by direct RNA sequencing (DRS). Bioinformatic analysis revealed 357 significant alterations in poly(A) site utilization that could be specifically linked to the αCP depletion. These APA events correlated strongly with the presence of C-rich sequences in close proximity to the impacted poly(A) addition sites. The most significant linkage was the presence of a C-rich motif within a window 30 to 40 bases 5′ to poly(A) signals (AAUAAA) that were repressed upon αCP depletion. This linkage is consistent with a general role for αCPs as enhancers of 3′ processing. These findings predict a role for αCPs in posttranscriptional control pathways that can alter the coding potential and/or levels of expression of subsets of mRNAs in the mammalian transcriptome.

For many mammalian genes, initiation of transcription during embryonic development must be subsequently sustained over extensive periods of adult life. It remains unclear whether maintenance of gene expression reflects the same set of pathways as are involved in initial gene activation. The human pituitary growth hormone (hGH-N) locus is activated in the differentiating somatotrope midway through embryogenesis by a multicomponent locus control region (LCR). DNase I-hypersensitive site I (HSI) of the LCR is essential to full developmental activation of the hGH-N locus. Here we demonstrate that conditional deletion of HSI from the active hGH locus in the adult pituitary effectively silences hGH-N expression. Analyses of chromatin structure and locus positioning demonstrate that a specific subset of the HSI functions active in the embryo retain their HSI dependence in the adult pituitary. These functions sustain engagement of the hGH locus with polymerase II (Pol II) factories, histone acetylation at the hGH-N promoter, and looping of the LCR to its target promoter. These data reveal that HSI is essential to both the maintenance and the initiation phases of gene expression. These observations contribute to our mechanistic understanding of how stable patterns of mammalian gene expression are established in a terminally differentiated cell.

Neurobeachin (NBEA), a brain-enriched multidomain scaffolding protein involved in neurotransmitter release and synaptic functioning, has been identified as a candidate gene for autism spectrum disorder (ASD) in four unrelated patients haploinsufficient for NBEA. The aim of this study was to map the behavioral phenotype of Nbea(+/-) mice in order to understand its contribution to the pathogenesis of ASD. ASD-like behavioral variables of Nbea(+/-) mice were related to basal neuronal activity in different brain regions by in situ hybridizations and extracellular field recordings of synaptic plasticity in hippocampal cornu ammonis 1 (CA1) region. Levels of BDNF and phosphorylated cAMP response element-binding protein (CREB) were measured in an attempt to investigate putatively underlying changes in these neuromolecules. Nbea(+/-) mice exhibit several ASD-like features, including changes in self-grooming behavior, social behaviors, conditioned fear responses, and spatial learning and memory, which coincided with enhanced long-term potentiation (LTP) in their CA1 region. The observed alterations in learning and memory and hippocampal LTP are concomitant with decreased expression of the immediate early gene zif268 in dorsomedial striatum and hippocampal CA1 region, increased CREB phosphorylation, and increased hippocampal BDNF expression. These findings indicate that Nbea haploinsufficiency leads to various molecular and cellular changes that affect neuroplasticity and behavioral functions in mice, and could thus underlie the ASD symptomatology in NBEA deficient humans. ispartof: Neurobiology Of Disease vol:51 pages:144-151 ispartof: location:United States status: published

Alternative splicing (AS) is a robust generator of mammalian transcriptome complexity. Splice site specification is controlled by interactions of cis-acting determinants on a transcript with specific RNA binding proteins. These interactions are frequently localized to the intronic U-rich polypyrimidine tracts (PPT) located 5' to the majority of splice acceptor junctions. αCPs (also referred to as polyC-binding proteins (PCBPs) and hnRNPEs) comprise a subset of KH-domain proteins with high affinity and specificity for C-rich polypyrimidine motifs. Here, we demonstrate that αCPs promote the splicing of a defined subset of cassette exons via binding to a C-rich subset of polypyrimidine tracts located 5' to the αCP-enhanced exonic segments. This enhancement of splice acceptor activity is linked to interactions of αCPs with the U2 snRNP complex and may be mediated by cooperative interactions with the canonical polypyrimidine tract binding protein, U2AF65. Analysis of αCP-targeted exons predicts a substantial impact on fundamental cell functions. These findings lead us to conclude that the αCPs play a direct and global role in modulating the splicing activity and inclusion of an array of cassette exons, thus driving a novel pathway of splice site regulation within the mammalian transcriptome.

The relationships of higher order chromatin organization to mammalian gene expression remain incompletely defined. The human Growth Hormone (hGH) multigene cluster contains five gene paralogs. These genes are selectively activated in either the pituitary or the placenta by distinct components of a remote locus control region (LCR). Prior studies have revealed that appropriate activation of the placental genes is dependent not only on the actions of the LCR, but also on the multigene composition of the cluster itself. Here, we demonstrate that the hGH LCR ‘loops’ over a distance of 28 kb in primary placental nuclei to make specific contacts with the promoters of the two GH genes in the cluster. This long-range interaction sequesters the GH genes from the three hCS genes which co-assemble into a tightly packed ‘hCS chromatin hub’. Elimination of the long-range looping, via specific deletion of the placental LCR components, triggers a dramatic disruption of the hCS chromatin hub. These data reveal a higher-order structural pathway by which long-range looping from an LCR impacts on local chromatin architecture that is linked to tissue-specific gene regulation within a multigene cluster.

Locus control regions (LCRs) comprise sets of DNA elements capable of establishing autonomous chromatin domains that support robust and physiologically appropriate expression of target genes, often working over extensive distances. Human growth hormone (hGH-N) expression in the pituitary is under the regulation of a well characterized LCR containing four DNase I hypersensitive sites (HSs). The two pituitary-specific HS, HSI and HSII, are located 14.5 and 15.5 kb 5' to the hGH-N promoter. HSI is essential for activation of hGH-N during pituitary development and for sustaining robust activity in the adult. To determine whether the closely linked HSII has a role in hGH-N expression, it was deleted from a previously validated hGH/P1 transgene. Analysis of three independent hGH/P1(ΔHSII) transgenic mouse lines revealed that this deletion had no adverse effect on the formation of HSI, yet resulted in a substantial loss (70%) in hGH-N mRNA expression. This loss of expression was accompanied by a corresponding reduction in recruitment of the pituitary-specific transcription factor Pit-1 to the hGH-N promoter and a selective decrease in promoter occupancy of the elongation-linked isoform of RNA polymerase II. Sufficiency of HSI and HSII in LCR activity was explored by establishing two additional sets of mouse transgenic lines in which DNA segments containing these HS were positioned within the λ phage genome. In this "neutral" DNA context, HSII was required for the recruitment of HAT activity. These data establish HSII as a nonredundant component of the hGH LCR essential for establishment of robust levels of hGH-N gene expression.

Insulin-like growth factor binding protein (IGFBP)-3 regulates cell proliferation and apoptosis in esophageal squamous cell carcinoma (ESCC) cells. We have investigated how the hypoxic tumor microenvironment in ESCC fosters the induction of IGFBP3. RNA interference experiments revealed that hypoxia-inducible factor (HIF)-1α, but not HIF-2α, regulates IGFBP3 mRNA induction. By chromatin immunoprecipitation and transfection assays, HIF-1α was found to transactivate IGFBP3 through a novel hypoxia responsive element (HRE) located at 57 kb upstream from the transcription start site. Metabolic labeling experiments demonstrated hypoxia-mediated inhibition of global protein synthesis. 7-Methyl GTP-cap binding assays suggested that hypoxia suppresses cap-dependent translation. Experiments using pharmacological inhibitors for mammalian target of rapamycin (mTOR) suggested that a relatively weak mTOR activity may be sufficient for cap-dependent translation of IGFBP3 under hypoxic conditions. Bicistronic RNA reporter transfection assays did not validate the possibility of an internal ribosome entry site as a potential mechanism for cap-independent translation for IGFBP3 mRNA. Finally, IGFBP3 mRNA was found enriched to the polysomes. In aggregate, our study establishes IGFBP3 as a direct HIF-1α target gene and that polysome enrichment of IGFBP3 mRNA may permit continuous translation under hypoxic conditions.—Natsuizaka, M., Naganuma, S., Kagawa, S., Ohashi, S., Ahmadi, A., Subramanian, H., Chang, S., Nakagawa, K. J., Ji, X., Liebhaber, S. A., Klein-Szanto, A. J., Nakagawa, H. Hypoxia induces IGFBP3 in esophageal squamous cancer cells through HIF-1α-mediated mRNA transcription and continuous protein synthesis.

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and rapidly degrades mRNAs containing premature termination codons (PTC). The strength of the NMD response appears to reflect multiple determinants on a target mRNA. We have previously reported that mRNAs containing PTCs in close proximity to the translation initiation codon (AUG-proximal PTCs) can substantially evade NMD. Here, we explore the mechanistic basis for this NMD resistance. We demonstrate that translation termination at an AUG-proximal PTC lacks the ribosome stalling that is evident in an NMD-sensitive PTC. This difference is associated with demonstrated interactions of the cytoplasmic poly(A)-binding protein 1, PABPC1, with the cap-binding complex subunit, eIF4G and the 40S recruitment factor eIF3 as well as the ribosome release factor, eRF3. These interactions, in combination, underlie critical 3'-5' linkage of translation initiation with efficient termination at the AUG-proximal PTC and contribute to an NMD-resistant PTC definition at an early phase of translation elongation.

Post-transcriptional controls are critical to gene regulation. These controls are frequently based on sequence-specific binding of trans-acting proteins to cis-acting motifs on target RNAs. Prior studies have revealed that the KH-domain protein, αCP, binds to a 3′ UTR C-rich motif of hα-globin mRNA and contributes to its cytoplasmic stability. Here, we report that this 3′ UTR αCP complex regulates the production of mature α-globin mRNA by enhancing 3′ processing of the hα-globin transcript. We go on to demonstrate that this nuclear activity reflects enhancement of both the cleavage and the polyadenylation reactions and that αCP interacts in vivo with core components of the 3′ processing complex. Consistent with its nuclear processing activity, our studies reveal that αCP assembles co-transcriptionally at the hα-globin chromatin locus and that this loading is selectively enriched at the 3′ terminus of the gene. The demonstrated linkage of nuclear processing with cytoplasmic stabilization via a common RNA–protein complex establishes a basis for integration of sequential controls critical to robust and sustained expression of a target mRNA.

Expression of mammalian GH is normally restricted to somatotropes and somatolactotropes (somatotrope lineages) in the anterior pituitary. The basis for this restriction remains incompletely understood. Recent studies indicate that deoxyribonuclease I hypersensitive site I (HSI) of the hGH locus control region, located at -14.5 kb relative to the hGH-N promoter, acts as a potent long-range enhancer of hGH-N transcription. Here we report that HSI is also critical to somatotrope-restriction of hGH-N expression. Loss of HSI activity, either by direct inactivation of HSI or by interference with HSI-dependent downstream events, results in a relaxation of hGH-N cell-type specification with expansion of hGH-N expression to the full spectrum of Pit-1 positive pituitary cell types. These findings expand the defined roles for HSI of the hGH locus control region to include somatotrope lineage restriction as well as transcriptional enhancement of hGH-N gene expression.

Post-transcriptional control of mRNA stability and translation is central to multiple developmental pathways. This control can be linked to cytoplasmic polyadenylation in certain settings. In maturing Xenopus oocytes, specific mRNAs are targeted for polyadenylation via recruitment of the Cytoplasmic Polyadenylation Element (CPE) binding protein (CPEB) to CPE(s) within the 3′ UTR. Cytoplasmic polyadenylation is also critical to early embryonic events, although corresponding determinants are less defined. Here, we demonstrate that the Xenopus ortholog of the poly(rC) binding protein αCP2 can recruit cytoplasmic poly(A) polymerase activity to mRNAs in Xenopus post-fertilization embryos, and that this recruitment relies on cis sequences recognized by αCP2. We find that the hα-globin 3′ UTR, a validated mammalian αCP2 target, constitutes an effective target for cytoplasmic polyadenylation in Xenopus embryos, but not during Xenopus oocyte maturation. We further demonstrate that the cytoplasmic polyadenylation activity is dependent on the action of the C-rich αCP-binding site in conjunction with the adjacent AAUAAA. Consistent with its ability to target mRNA for poly(A) addition, we find that XαCP2 associates with core components of the Xenopus cytoplasmic polyadenylation complex, including the cytoplasmic poly(A) polymerase XGLD2. Furthermore, we observe that the C-rich αCP-binding site can robustly enhance the activity of a weak canonical oocyte maturation CPE in early embryos, possibly via a direct interaction between XαCP2 and CPEB1. These studies establish XαCP2 as a novel cytoplasmic polyadenylation trans factor, indicate that C-rich sequences can function as noncanonical cytoplasmic polyadenylation elements, and expand our understanding of the complexities underlying cytoplasmic polyadenylation in specific developmental settings.

Posttranscriptional regulation is of critical importance during mammalian spermiogenesis. A set of mRNAs that encode proteins critical to normal sperm formation are synthesized early in the process of male germ cell differentiation and are stored in a repressed state. These mRNAs are subsequently translationally activated during the process of spermatid elongation and maturation. Of note, the translationally repressed mRNAs contain long poly(A) tails that are dramatically shortened during the translational activation process. Understanding the mechanisms that underlie this process of mRNA storage and subsequent translational activation has been a long-standing goal. The relationship of the poly(A) tail to translational control is intimately related to the functions of the cognate poly(A)-binding proteins (PABPs). In this issue of the JCI, Yanagiya and colleagues use a set of knockout mice to demonstrate a novel functional role for a particular modulator of PABP function, PABP-interacting protein 2a (PAIP2A), in the normal terminal differentiation of male germ cells.

The alpha-globin poly(C)-binding proteins (alphaCPs) comprise an abundant and widely expressed set of K-homolog domain RNA-binding proteins. alphaCPs regulate the expression of a number of cellular and viral mRNAs at the levels of splicing, stability, and translation. Previous surveys have identified 160 mRNAs that are bound by alphaCP in the human hematopoietic cell line, K562. To explore the functions of these alphaCP/mRNA interactions, we identified mRNAs whose levels are altered in K562 cells acutely depleted of the two major alphaCP proteins, alphaCP1 and alphaCP2. Microarray analysis identified 27 mRNAs that are down-regulated and 14 mRNAs that are up-regulated in the alphaCP1/2-co-depleted cells. This alphaCP1/2 co-depletion was also noted to inhibit cell proliferation and trigger a G(1) cell cycle arrest. Targeted analysis of genes involved in cell cycle control revealed a marked increase in p21(WAF) mRNA and protein. Analysis of mRNP complexes in K562 cells demonstrates in vivo association of p21(WAF) mRNA with alphaCP1 and alphaCP2. In vitro binding assays indicate that a 127-nucleotide region of the 3'-untranslated region of p21(WAF) interacts with both alphaCP1 and alphaCP2, and co-depletion of alphaCP1/2 results in a marked increase in p21(WAF) mRNA half-life. p21(WAF) induction and G(1) arrest in the alphaCP1/2-co-depleted cells occur in the absence of p53 and are not observed in cells depleted of the individual alphaCP isoforms. The apparent redundancy in the actions of alphaCP1 and alphaCP2 upon p21(WAF) expression correlates with a parallel redundancy in their effects on cell cycle control. These data reveal a pivotal role for alphaCP1 and alphaCP2 in a p53-independent pathway of p21(WAF) control and cell cycle progression.

Exposure of cells to endoplasmic reticulum (ER) stress leads to activation of phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway and transcriptional induction of the inhibitor of apoptosis family of proteins. One of the proximal effectors of the ER stress response, the PKR-like ER kinase (PERK), leads to cellular adaptation to stress by multiple mechanisms, including attenuation of protein synthesis and transcriptional induction of pro-survival genes. Although PERK activity leads to cellular adaptation to ER stress, we now demonstrate that PERK activity also inhibits the ER stress-induced apoptotic program through the induction of cellular inhibitor of apoptosis (cIAP1 and cIAP2) proteins. This induction of IAPs occurs through both transcriptional and translational responses that are PERK dependent. Reintroduction of cIAP1 or cIAP2 expression into PERK-/- murine embryonic fibroblasts during ER stress delays the early onset of ER stress-induced caspase activation and apoptosis observed in these cells. Furthermore, we demonstrate that the activation of the PI3K-Akt pathway by ER stress is dependent on PERK, suggesting additional ways in which PERK activity protects cells from ER stress-induced apoptosis.

Antisense oligodeoxynucleotides (AONs) and short interfering RNAs (siRNAs) effect posttranscriptional gene silencing (PTGS) by hybridizing to an mRNA and then directing its cleavage. To understand the constraints that mRNA structure imposes on AON- vs. siRNA-mediated PTGS, AON- and siRNA-mediated cleavage of defined mRNA structures was monitored in Drosophila embryo whole-cell lysates. We observed that AON-directed cleavage was ≈3-fold faster than cleavage with a siRNA directed to the same target site. Furthermore, and unexpectedly, AON-mediated cleavage was found to be much less fastidious with respect to target sequence accessibility, as measured by the presence of unpaired nucleotides, than a corresponding siRNA. Nonetheless, in vivo , siRNAs silenced their mRNA target at least 2-fold more efficiently than the corresponding AON. These seemingly contradictory results suggested that additional, as yet undefined factors play an important role in regulating PTGS efficiency in vivo . We used a well defined RNA-binding protein, αCP, and its corresponding high-affinity RNA-binding site to explore this hypothesis. We found that prebound αCP effectively blocked AON-mediated cleavage of the RNA-binding site compared with cleavage of the site in the absence of αCP. We conclude that higher-order structures formed by RNA and bound proteins play an important role in determining the efficiency of AON-directed PTGS. We hypothesize that strategies aimed at removing RNA-binding proteins might significantly improve AON-mediated PTGS in vivo .

Nonlinear chromatin configurations can juxtapose widely separated elements within a genomic locus; however, it remains unclear how these structures are established and contribute to transcriptional control. A 5′‐remote locus control region (LCR) regulates the human growth hormone ( hGH‐N ) gene. HSI, a pituitary‐specific component of the hGH LCR, establishes a domain of polymerase II (PolII) transcription 5′ to hGH‐N . Repression of this transcriptional domain by HSI deletion or PolII blockade decreases hGH‐N expression. Here, we show that hGH‐N activation is accompanied by positioning of the hGH‐N promoter to this LCR transcriptional domain. Selectively blocking LCR transcription inhibits the formation of this active ‘looped’ conformation. Thus, HSI is crucial for establishing a domain of noncoding PolII transcription, and this domain is intimately linked with chromatin organization of the active hGH‐N locus. This integration of LCR transcription with chromatin reconfiguration constitutes a robust pathway for long‐range gene activation.

mRNA surveillance pathways selectively clear defective mRNAs from the cell. As such, these pathways serve as important modifiers of genetic disorders. Nonsense-mediated decay (NMD), the most intensively studied surveillance pathway, recognizes mRNAs with premature termination codons (PTCs). In mammalian systems the location of a PTC more than 50 nucleotides 5′ to the terminal exon–exon junction is a critical determinant of NMD. However, mRNAs with nonsense codons that fulfill this requirement but are located very early in the open reading frame can effectively evade NMD. The unexpected resistance of such mRNAs with AUG-proximal PTCs to accelerated decay suggests that important determinants of NMD remain to be identified. Here, we report that an NMD-sensitive mRNA can be stabilized by artificially tethering the cytoplasmic poly(A) binding protein 1, PABPC1, at a PTC-proximal position. Remarkably, the data further suggest that NMD of an mRNA with an AUG-proximal PTC can also be repressed by PABPC1, which might be brought into proximity with the PTC during cap-dependent translation and 43S scanning. These results reveal a novel parameter of NMD in mammalian cells that can account for the stability of mRNAs with AUG-proximal PTCs. These findings serve to expand current mechanistic models of NMD and mRNA translation.

The somatic hypermutational (SHM) status of the immunoglobulin heavy-chain variable (IgVH) gene is a powerful prognostic factor in patients with chronic lymphocytic leukemia (CLL). However, IgVH SHM analysis is not well-suited to routine use in the clinical diagnostic laboratory. ZAP70 expression is a potential surrogate for the absence of SHM. Given the current problems with the standardization of ZAP70 assessment by flow cytometry, we sought an alternative approach, using immunohistochemistry (IHC). The utility of IHC is largely restricted to tissues, precluding its routine application to most patients with CLL who are typically diagnosed based upon peripheral blood (PB) findings. Accordingly, we developed an IHC assay that can be performed on PB. Enriched PB mononuclear cells from 29 patients with CLL were analyzed for ZAP70 expression by IHC on paraffin-embedded cell blocks, using standard techniques. IgVH SHM analysis was performed on all cases, and clinical features recorded. Seventeen specimens (59%) were negative for ZAP70 expression and 12 (41%) were positive for ZAP70 expression. SHM was evident in 20 specimens (69%), and absent in 9 (31%). Seventy-six percent of the specimens (22/29) displayed "concordant" ZAP70 and SHM results, in that 15 (52%) were SHM-positive/ZAP70 negative, whereas 7 (24%) were SHM-negative/ZAP70 positive. ZAP70 expression in this small cohort correlated with poor clinical outcome. Importantly, IHC analysis of ZAP70 in PB is a simple, reliable, robust assay that may have a valuable role in the routine clinical laboratory assessment of patients with CLL.

The hGH cluster contains a single human pituitary growth hormone gene (hGH-N) and four placenta-specific paralogs. Activation of the cluster in both tissues depends on 5' remote regulatory elements. The pituitary-specific locus control elements DNase I-hypersensitive site I (HSI) and HSII, located 14.5 kb 5' of the cluster (position -14.5), establish a continuous domain of histone acetylation that extends to and activates hGH-N in the pituitary gland. In contrast, histone modifications in placental chromatin are restricted to the more 5'-remote HSV-HSIII region (kb -28 to -32) and to the placentally expressed genes in the cluster, with minimal modification between these two regions. These data predict distinct modes of hGH cluster gene activation in the pituitary and placenta. Here we used cell culture models to track structural changes at the hGH locus through placental-gene activation. The data revealed that this process was initiated in primary cytotrophoblasts by histone H3K4 di- and trimethylation and H4 acetylation restricted to HSV and to the individual placental-gene repeat (PGR) units within the cluster. Later stages of transcriptional induction were accompanied by enhancement and extension of these modifications and by robust H3 acetylation at HSV, at HSIII, and throughout the placental-gene regions. These data suggested that elements restricted to HSIII-HSV regions and each individual PGR might be sufficient for activation of the hCS genes. This model was tested by comparing hCS transgene expression in the placentas of mouse embryos carrying a full hGH cluster to that in placentas in which the HSIII-HSV region was directly linked to the individual hCS-A PGR unit. The findings indicate that the HSIII-HSV region and the PGR units, although targeted for initial chromatin structural modifications, are insufficient to activate gene expression and that this process is dependent on additional, as-yet-unidentified chromatin determinants.

Activation of eukaryotic genes often relies on remote chromatin determinants. How these determinants function remains poorly understood. The hGH gene is activated by a 5'-remote locus control region (LCR). Pituitary-specific DNase I hypersensitive site I (HSI), the dominant hGH LCR element, is separated from the hGH-N promoter by a 14.5 kb span that encompasses the B-lymphocyte-specific CD79b gene. Here, we describe a domain of noncoding Pol II transcription in pituitary somatotropes that includes the hGH LCR and adjacent CD79b locus. This entire "LCR domain of transcription" is HSI [corrected] dependent and terminates 3' to CD79b, leaving a gap in transcription between this domain and the target hGH-N promoter. Insertion of a Pol II terminator within the LCR blocks CD79b transcription and represses hGH-N expression. These data document an essential role for LCR transcription in long-range control, link "bystander"CD79b transcription to this process, and support a unique model for locus activation.

Random assortment within mammalian genomes juxtaposes genes with distinct expression profiles. This organization, along with the prevalence of long-range regulatory controls, generates a potential for aberrant transcriptional interactions. The human CD79b/GH locus contains six tightly linked genes with three mutually exclusive tissue specificities and interdigitated control elements. One consequence of this compact organization is that the pituitary cell-specific transcriptional events that activate hGH-N also trigger ectopic activation of CD79b. However, the B-cell-specific events that activate CD79b do not trigger reciprocal activation of hGH-N. Here we utilized DNase I hypersensitive site mapping, chromatin immunoprecipitation, and transgenic models to explore the basis for this asymmetric relationship. The results reveal tissue-specific patterns of chromatin structures and transcriptional controls at the CD79b/GH locus in B cells distinct from those in the pituitary gland and placenta. These three unique transcriptional environments suggest a set of corresponding gene expression pathways and transcriptional interactions that are likely to be found juxtaposed at multiple sites within the eukaryotic genome.

The human vitamin D-binding protein (hDBP) gene is a member of a cluster that includes albumin, alpha-fetoprotein and alpha-albumin genes. The common origin, physical linkage and hepatic expression of these four genes predict shared regulatory element(s). However, separation of hDBP from the other three genes by 1.5 Mb argues that hDBP may be under autonomous control. To test for hDBP autonomy, mouse lines were generated with a transgene containing the hDBP gene along with extensive flanking sequences. Expression of this transgene was hepatic, robust and proportional to transgene copy number. DNase I hypersensitive site (HS) mapping revealed five liver-specific HS at the hDBP locus: HSI and HSIII at -2.1 kb and -0.13 kb upstream of the transcription initiation site, HSIV and HSV within intron 1 and HSVII located 3' to the poly(A) site. A second transgene with minimal flanking sequences confirmed the sufficiency of these gene-proximal determinants for hepatic activation. The hepatic-specific HS aligned with segments of phylogenetically conserved non-coding sequences. These data demonstrate the autonomy of the hDBP locus and suggest that this control is mediated by chromatin-based locus control determinants in close proximity to, and within the transcription unit.

mRNA stability is a critical determinant of normal red blood cell development and function. The long half-life of globin mRNA is central to the continued synthesis of globin proteins throughout all stages of erythropoiesis, even as the cells undergo programmed transcriptional arrest during terminal differentiation. Studies of a naturally occurring alpha-thalassemic mutation that triggers marked destabilization of alpha-globin mRNA first led investigators to search for a stability determinant in the 3'-untranslated region (3'UTR). Analysis of this region identified three cytosine-rich (C-rich) segments that contributed to alpha-globin mRNA stability when studied in transfected erythroid cells. Subsequently, in vitro studies demonstrated assembly of a sequence-specific ribonucleic-protein (RNP) complex at this site. Mutations in the 3'UTR that blocked formation of this "alpha-complex" in vitro resulted in a parallel destabilization of alpha-globin mRNA in transfected cells. Members of the alpha-globin poly(C)-binding protein (alphaCP) subfamily of heteronuclear (hn) RNP K homology (KH) domain RNA-binding proteins have been identified as essential protein components of the alpha-complex. In vitro studies suggested that additional proteins may also contribute to alpha-complex structure and/or function. Surveys of additional highly stable mRNAs point to a general function for the alpha-complex in mRNA stabilization. In vitro and in vivo analyses indicated that the alpha-complex stabilizes alpha-globin mRNA by two mechanisms: control of 3'-terminal deadenylation and steric protection of an endoribonuclease-sensitive site. Confirmation of these pathways, determination of their relative importance, and generalization of these findings to additional systems await future studies.

Previous studies suggest that high-level stability of a subset of mammalian mRNAs is linked to a C-rich motif in the 3' untranslated region (3'UTR). High-level expression of human alpha-globin mRNA (h alpha-globin mRNA) in erythroid cells has been specifically attributed to formation of an RNA-protein complex comprised of a 3'UTR C-rich motif and an associated 39-kDa poly(C) binding protein, alpha CP. Documentation of this RNA-protein alpha-complex has been limited to in vitro binding studies, and its impact has been monitored by alterations in steady-state mRNA. Here we demonstrate that alpha CP is stably bound to h alpha-globin mRNA in vivo, that alpha-complex assembly on the h alpha-globin mRNA is restricted to the 3'UTR C-rich motif, and that alpha-complex assembly extends the physical half-life of h alpha-globin mRNA selectively in erythroid cells. Significantly, these studies also reveal that an artificially tethered alpha CP has the same mRNA-stabilizing activity as the native alpha-complex. These data demonstrate a unique contribution of the alpha-complex to h alpha-globin mRNA stability and support a model in which the sole function of the C-rich motif is to selectively tether alpha CP to a subset of mRNAs. Once bound, alpha CP appears to be fully sufficient to trigger downstream events in the stabilization pathway.

Posttranscriptional controls play a major role in eucaryotic gene expression. These controls are mediated by sequence-specific interactions of cis-acting determinants in target mRNAs with one or more protein factors. The positioning of a subset of these mRNA-protein (RNP) complexes within the 3' untranslated region (3' UTR) may allow them to remain associated with the mRNA during active translation. Robust expression of human alpha-globin mRNA during erythroid differentiation has been linked to formation of a binary complex between a KH-domain protein, alphaCP, and a 3' UTR C-rich motif. Detection of this "alpha-complex" has been limited to in vitro studies, and the functional state of the alpha-globin mRNA targeted by alphaCP has not been defined. In the present study we demonstrate that a significant fraction of alphaCP is associated with polysomal mRNA. Targeted analysis of the polysomal RNP complexes revealed that alphaCP is specifically bound to actively translating alpha-globin mRNA. The bound alphaCP is restricted to the poly(C)-rich 3' UTR motif and is dislodged when ribosomes are allowed to enter this region. These data validate the general importance of the 3' UTR as a sheltered site for RNP complexes and support a specific model in which the stabilizing function of alphaCP is mediated on actively translating target mRNAs.

RNA-binding proteins are involved in the regulation of many aspects of eukaryotic gene expression. Targeted interference with RNA-protein interactions could offer novel approaches to modulation of expression profiles, alteration of developmental pathways, and reversal of certain disease processes. Here we investigate a decoy strategy for the study of the alphaCP subgroup of KH-domain RNA-binding proteins. These poly(C)-binding proteins have been implicated in a wide spectrum of posttranscriptional controls. Three categories of RNA decoys to alphaCPs were studied: poly(C) homopolymers, native mRNA-binding sites, and a high-affinity structure selected from a combinatorial library. Native chemistry was found to be essential for alphaCP decoy action. Because alphaCP proteins are found in both the nucleus and cytoplasm, decoy cassettes were incorporated within both nuclear (U1 snRNA) and cytoplasmic (VA1 RNA) RNA frameworks. Several sequences demonstrated optimal decoy properties when assayed for protein-binding and decoy bioactivity in vitro. A subset of these transcripts was shown to mediate targeted inhibition of alphaCP-dependent translation when expressed in either the nucleus or cytoplasm of transfected cells. Significantly, these studies establish the feasibility of developing RNA decoys that can selectively target biologic functions of abundant and widely expressed RNA binding proteins.

The poly(C) binding proteins (PCBPs) are encoded at five dispersed loci in the mouse and human genomes. These proteins, which can be divided into two groups, hnRNPs K/J and the alphaCPs (alphaCP1-4), are linked by a common evolutionary history, a shared triple KH domain configuration, and by their poly(C) binding specificity. Given these conserved characteristics it is remarkable to find a substantial diversity in PCBP functions. The roles of these proteins in mRNA stabilization, translational activation, and translational silencing suggest a complex and diverse set of post-transcriptional control pathways. Their additional putative functions in transcriptional control and as structural components of important DNA-protein complexes further support their remarkable structural and functional versatility. Clearly the identification of additional binding targets and delineation of corresponding control mechanisms and effector pathways will establish highly informative models for further exploration.

Gene activation in higher eukaryotes is often under the control of regulatory elements quite distant from their target promoters. It is unclear how such long-range control is mediated. Here we show that a single determinant of the human growth hormone locus control region (hGH LCR) located 14.5 kb 5prime prime or minute to the hGH-N promoter has a critical, specific, and nonredundant role in facilitating promoter trans factor binding and activating hGH-N transcription. Significantly, this same determinant plays an essential role in establishing a 32 kb acetylated domain that encompasses the entire hGH LCR and the contiguous hGH-N promoter. These data support a model for long-range gene activation via LCR-mediated targeting and extensive spreading of core histone acetylation.