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Determining the mechanisms by which genes are switched on and off during development and differentiation is a key aim of current biomedical research. Gene transcription has been widely observed to occur in a discontinuous fashion, with short bursts of activity interspersed with longer periods of inactivity. It is currently not known if or how this dynamic behaviour changes as mammalian cells differentiate. To investigate this, using a newly developed on-microscope analysis, we monitored mouse α-globin transcription in live cells throughout sequential stages of erythropoiesis. We find that changes in the overall levels of α-globin transcription are most closely associated with changes in the fraction of time a gene spends in the active transcriptional state. We identify differences in the patterns of transcriptional bursting throughout differentiation, with maximal transcriptional activity occurring in the mid-phase of differentiation. Early in differentiation, we observe increased fluctuation in the patterns of transcriptional activity whereas at the peak of gene expression, in early and intermediate erythroblasts, transcription appears to be relatively stable and efficient. Later during differentiation as α-globin expression declines, we again observed more variability in transcription within individual cells. We propose that the observed changes in transcriptional behaviour may reflect changes in the stability of enhancer-promoter interactions and the formation of active transcriptional compartments as gene expression is turned on and subsequently declines at sequential stages of differentiation.

The α- and β-globin loci harbor developmentally expressed genes, which are silenced throughout post-natal life. Reactivation of these genes may offer therapeutic approaches for the hemoglobinopathies, the most common single gene disorders. Here, we address mechanisms regulating the embryonically expressed α-like globin, termed ζ-globin. We show that in embryonic erythroid cells, the ζ-gene lies within a ~65 kb sub-TAD (topologically associating domain) of open, acetylated chromatin and interacts with the α-globin super-enhancer. By contrast, in adult erythroid cells, the ζ-gene is packaged within a small (~10 kb) sub-domain of hypoacetylated, facultative heterochromatin within the acetylated sub-TAD and that it no longer interacts with its enhancers. The ζ-gene can be partially re-activated by acetylation and inhibition of histone de-acetylases. In addition to suggesting therapies for severe α-thalassemia, these findings illustrate the general principles by which reactivation of developmental genes may rescue abnormalities arising from mutations in their adult paralogues., Globin loci harbor genes that are expressed embryonically and silenced postnatally. Here the authors show that zeta-globin silencing depends upon selective hypoacetylation of its TAD subdomain, which blocks its interaction with the alpha-globin super-enhancer, and zeta-globin can be reactivated by acetylation.

Mammalian genomes are subdivided into large (50-2000 kb) regions of chromatin referred to as Topologically Associating Domains (TADs or sub-TADs). Chromatin within an individual TAD contacts itself more frequently than with regions in surrounding TADs thereby directing enhancer-promoter interactions. In many cases, the borders of TADs are defined by convergently orientated boundary elements associated with CCCTC-binding factor (CTCF), which stabilises the cohesin complex on chromatin and prevents its translocation. This delimits chromatin loop extrusion which is thought to underlie the formation of TADs. However, not all CTCF-bound sites act as boundaries and, importantly, not all TADs are flanked by convergent CTCF sites. Here, we examined the CTCF binding sites within a ∼70 kb sub-TAD containing the duplicated mouse α-like globin genes and their five enhancers (5’-R1-R2-R3-Rm-R4-α1-α2-3’). The 5’ border of this sub-TAD is defined by a pair of CTCF sites. Surprisingly, we show that deletion of the CTCF binding sites within and downstream of the α-globin locus leaves the sub-TAD largely intact. The predominant 3’ border of the sub-TAD is defined by a steep reduction in contacts: this corresponds to the transcribed α2-globin gene rather than the CTCF sites at the 3’-end of the sub-TAD. Of interest, the almost identical α1- and α2-globin genes interact differently with the enhancers, resulting in preferential expression of the proximal α1-globin gene which behaves as a partial boundary between the enhancers and the distal α2-globin gene. Together, these observations provide direct evidence that actively transcribed genes can behave as boundary elements.Significance StatementMammalian genomes are complex, organised 3D structures, partitioned into Topologically Associating Domains (TADs): chromatin regions that preferentially self-interact. These chromatin interactions are thought to be driven by a mechanism that continuously extrudes chromatin loops, forming structures delimited by chromatin boundary elements and reflecting the activity of enhancers and promoters. Boundary elements bind architectural proteins such as CCCTC-binding factor (CTCF). Previously, an overlap between the functional roles of enhancers and promoters has been shown. However, whether there is overlap between enhancers/promoters and boundary elements is not known. Here, we show that actively transcribed genes can also behave as boundary elements, similar to CTCF boundaries. In both cases, multi-protein complexes bound to these regions may stall the process of chromatin loop extrusion.

The genome is organized via CTCF–cohesin-binding sites, which partition chromosomes into 1–5 megabase (Mb) topologically associated domains (TADs), and further into smaller sub-domains (sub-TADs). Here we examined in vivo an ∼80 kb sub-TAD, containing the mouse α-globin gene cluster, lying within a ∼1 Mb TAD. We find that the sub-TAD is flanked by predominantly convergent CTCF–cohesin sites that are ubiquitously bound by CTCF but only interact during erythropoiesis, defining a self-interacting erythroid compartment. Whereas the α-globin regulatory elements normally act solely on promoters downstream of the enhancers, removal of a conserved upstream CTCF–cohesin boundary extends the sub-TAD to adjacent upstream CTCF–cohesin-binding sites. The α-globin enhancers now interact with the flanking chromatin, upregulating expression of genes within this extended sub-TAD. Rather than acting solely as a barrier to chromatin modification, CTCF–cohesin boundaries in this sub-TAD delimit the region of chromatin to which enhancers have access and within which they interact with receptive promoters. Hanssen et al. show that CTCF–cohesin binding sites at the α-globin gene cluster function as boundaries to restrict the interaction of enhancers with the flanking chromatin, thus preventing abnormal gene expression.

Gene transcription occurs via a cycle of linked events including initiation, promoter proximal pausing and elongation of RNA polymerase II (Pol II). A key question is how do transcriptional enhancers influence these events to control gene expression? Here we have used a new approach to quantify transcriptional initiation and pausing in vivo, while simultaneously identifying transcription start sites (TSSs) and pause-sites (TPSs) from single RNA molecules. When analyzed in parallel with nascent RNA-seq, these data show that differential gene expression is achieved predominantly via changes in transcription initiation rather than Pol II pausing. Using genetically engineered mouse models deleted for specific enhancers we show that these elements control gene expression via Pol II recruitment and/or initiation rather than via promoter proximal pause release. Together, our data show that enhancers, in general, control gene expression predominantly by Pol II recruitment and initiation rather than via pausing.

Gene transcription occurs via a cycle of linked events including initiation, promoter proximal pausing and elongation of RNA polymerase II (Pol II). A key question is how do transcriptional enhancers influence these events to control gene expression? Here we have used a new approach to quantify transcriptional initiation and pausing in vivo, while simultaneously identifying transcription start sites (TSSs) and pause-sites (TPSs) from single RNA molecules. When analyzed in parallel with nascent RNA-seq, these data show that differential gene expression is achieved predominantly via changes in transcription initiation rather than Pol II pausing. Using genetically engineered mouse models deleted for specific enhancers we show that these elements control gene expression via Pol II recruitment and/or initiation rather than via promoter proximal pause release. Together, our data show that enhancers, in general, control gene expression predominantly by Pol II recruitment and initiation rather than via pausing.

ATRX is a chromatin remodelling factor found at a wide range of tandemly repeated sequences including telomeres (TTAGGG) n . ATRX mutations are found in nearly all tumours that maintain their telomeres via the alternative lengthening of telomere (ALT) pathway, and ATRX is known to suppress this pathway. Here, we show that recruitment of ATRX to telomeric repeats depends on repeat number, orientation and, critically, on repeat transcription. Importantly, the transcribed telomeric repeats form RNA–DNA hybrids (R‐loops) whose abundance correlates with the recruitment of ATRX. Here, we show loss of ATRX is also associated with increased R‐loop formation. Our data suggest that the presence of ATRX at telomeres may have a central role in suppressing deleterious DNA secondary structures that form at transcribed telomeric repeats, and this may account for the increased DNA damage, stalling of replication and homology‐directed repair previously observed upon loss of ATRX function.

We have analysed the effect of a 1.4 kb segment of DNA containing the upstream alpha globin regulatory element (HS-40) on human alpha globin gene expression in fetal mice and lines of transgenic mice. High levels of tissue-specific, human alpha mRNA expression were seen in all transgenic animals and in this sense expression was position independent. However, the level of human alpha mRNA expression per integrated gene copy decreased during development and was inversely related to copy number. The limitation in expression with increasing gene copy number was shown to be in cis since homozygotes for the transgene produced twice as much human alpha mRNA as hemizygotes. In many respects HS -40 appears similar to single elements within the previously described beta globin locus control region and in cross breeding experiments we have shown that HS -40 behaves in a similar manner to such elements in transgenic mice.

Previous work has suggested that the promoter regions of the human embryonic zeta 2 and epsilon globin genes contain negative regulatory regions that could play a role in the repression of these genes in postembryonic erythroblasts. We have examined this possibility by studying the expression of these genes in mouse erythroleukemia cells, an adult erythroid cell line that might be expected to contain repressor molecules that would bind to the putative negative regulatory regions. When attached to appropriate upstream regulatory elements (alpha HS-40 and beta HS1,2) both the zeta and epsilon genes were expressed in these cells at a low level, but no increase in expression was observed when similar constructs lacking the proposed negative regulatory sequences were introduced into these cells. These results cast doubt on the possibility that these sequences play a major role in the developmental repression of the embryonic globin genes, unless they function only in a normal chromosomal organization.

A substantial amount of organismal complexity is thought to be encoded by enhancers which specify the location, timing, and levels of gene expression. In mammals there are more enhancers than promoters which are distributed both between and within genes. Here we show that activated, intragenic enhancers frequently act as alternative tissue-specific promoters producing a class of abundant, spliced, multiexonic poly(A) + RNAs (meRNAs) which reflect the host gene's structure. meRNAs make a substantial and unanticipated contribution to the complexity of the transcriptome, appearing as alternative isoforms of the host gene. The low protein-coding potential of meRNAs suggests that many meRNAs may be byproducts of enhancer activation or underlie as-yet-unidentified RNA-encoded functions. Distinguishing between meRNAs and mRNAs will transform our interpretation of dynamic changes in transcription both at the level of individual genes and of the genome as a whole. © 2012 Elsevier Inc..

Erythroid-specific DNase 1 hypersensitive sites have been identified at the promoters of the human alpha-like genes and within the region from 4 to 40 kb upstream of the gene cluster. One of these sites, HS-40, has been shown previously to be the major regulator of tissue-specific alpha-globin gene expression. We have now examined the function of other hypersensitive sites by studying the expression in mouse erythroleukemia (MEL) cells of various fragments containing these sites attached to HS-40 and an alpha-globin gene. High level expression of the alpha gene was observed in all cases. When clones of MEL cells bearing a single copy of the alpha-globin gene fragments were examined, expression levels were similar to those of the endogenous mouse alpha genes and similar to MEL cells bearing beta gene constructs under the control of the beta-globin locus control region. However, there was no evidence that the additional hypersensitive sites increased the level of expression or conferred copy number dependence on the expression of a linked alpha gene in MEL cells.

The Congenital Dyseyrthropoetic Anemias (CDA) are a heterogeneous group of inborn defects characterized by anemia of varying severity and morphological abnormalities of bone marrow erythroblasts. Studying their pathology has the potential to contribute to the understanding of the normal process of erythropoiesis. In CDA type I, the phenotype is largely limited to the red cell lineage with erythroblasts showing characteristic spongy nuclear chromatin on electron microscopy. Its genetic basis is due to missense mutations in the CDAN1 gene (chromosome 15q15), but the biological function of the highly conserved and non-redundant codanin-1 protein remains entirely unknown. We have produced a mouse model for CDA type I by generating transgenic mice from an ES cell line carrying a gene-trap (splice acceptor/β-galactosidase gene/neomycin resistance gene) in intron 25 of the murine CDAN1 gene. Blood and bone marrow from CDAN1gt/wt heterozygotes is morphologically and quantitatively indistinguishable from wild type animals. Northern blot analysis and RT-qPCR of codanin-1 RNA expression confirms a broad pattern of expression with the highest levels seen in erythroid tissue. Flow-cytometric analysis of β-galactosidase activity in erythroid cells from phenylhydrazine treated adult spleens shows the highest levels in the CD71high/Ter119low early erythroblasts, declining with further erythroid maturation. Furthermore, indirect immunofluorescence localizes the codanin-1/β-galactosidase fusion product in the nucleus of the CD71high/Ter119low early erythroblasts, particularly at the interface between euchromatin and heterochromatin. The presence of an in-frame β-gal in the gene trap insert, therefore, provides a method to analyze gene and protein expression from the CDAN1 promoter, and additional distribution data from heterozygous embryos and adults will be presented. Heterozygote crosses produced no homozygotes among liveborn progeny, suggesting embryonic lethality of this state. Analysis of embryos at different stages suggests development of homozygotes ceased at ~6.5d of gestation, prior to the onset of erythropoiesis. These results from the first transgenic mouse model for CDA type I, highlight a non-erythroid role for codanin-1 in early embryonic development, in addition to its role in adult human erythropoiesis. The embryonic lethality of the mouse model is consistent with the absence of homozygote null mutations in the CDAN1 genes analyzed so far in human patients. Creation of mice with the CDA type I phenotype may have to await a knock-in transgenic containing a human mutation, which is currently in preparation. The model described here should be valuable for further studies of the intracellular localization of codanin-1 and the identification of any relevant protein binding partners.

© 2016 Nature America, Inc. All rights reserved. Many genes determining cell identity are regulated by clusters of Mediator-bound enhancer elements collectively referred to as super-enhancers. These super-enhancers have been proposed to manifest higher-order properties important in development and disease. Here we report a comprehensive functional dissection of one of the strongest putative super-enhancers in erythroid cells. By generating a series of mouse models, deleting each of the five regulatory elements of the α-globin super-enhancer individually and in informative combinations, we demonstrate that each constituent enhancer seems to act independently and in an additive fashion with respect to hematological phenotype, gene expression, chromatin structure and chromosome conformation, without clear evidence of synergistic or higher-order effects. Our study highlights the importance of functional genetic analyses for the identification of new concepts in transcriptional regulation.

The role of DNA sequence in determining chromatin state is incompletely understood. We have previously demonstrated that large chromosomal segments from human cells recapitulate their native chromatin state in mouse cells, but the relative contribution of local sequences versus their genomic context remains unknown. In this study, we compare orthologous chromosomal regions for which the human locus establishes prominent sites of Polycomb complex recruitment in pluripotent stem cells, whereas the corresponding mouse locus does not. Using recombination-mediated cassette exchange at the mouse locus, we establish the primacy of local sequences in the encoding of chromatin state. We show that the signal for chromatin bivalency is redundantly encoded across a bivalent domain and that this reflects competition between Polycomb complex recruitment and transcriptional activation. Furthermore, our results suggest that a high density of unmethylated CpG dinucleotides is sufficient for vertebrate Polycomb recruitment. This model is supported by analysis of DNA methyltransferase-deficient embryonic stem cells.

Muenke syndrome, defined by heterozygosity for a Pro250Arg substitution in fibroblast growth factor receptor 3 (FGFR3), is the most common genetic cause of craniosynostosis in humans. We have used gene targeting to introduce the Muenke syndrome mutation (equivalent to P244R) into the murine Fgfr3 gene. A rounded skull and shortened snout (often skewed) with dental malocclusion was observed in a minority of heterozygotes and many homozygotes. Development of this incompletely penetrant skull phenotype was dependent on genetic background and sex, with males more often affected. However, these cranial abnormalities were rarely attributable to craniosynostosis, which was only present in 2/364 mutants; more commonly, we found fusion of the premaxillary and/or zygomatic sutures. We also found decreased cortical thickness and bone mineral densities in long bones. We conclude that although both cranial and long bone development is variably affected by the murine Fgfr3(P244R) mutation, coronal craniosynostosis is not reliably reproduced.

To address the mechanism by which the human globin genes are activated during erythropoiesis, we have used a tiled microarray to analyze the pattern of transcription factor binding and associated histone modifications across the telomeric region of human chromosome 16 in primary erythroid and nonerythroid cells. This 220-kb region includes the α globin genes and 9 widely expressed genes flanking the α globin locus. This un-biased, comprehensive analysis of transcription factor binding and histone modifications (acetylation and methylation) described here not only identified all known cis-acting regulatory elements in the human α globin cluster but also demonstrated that there are no additional erythroid-specific regulatory elements in the 220-kb region tested. In addition, the pattern of histone modification distinguished promoter elements from potential enhancer elements across this region. Finally, comparison of the human and mouse orthologous regions in a unique mouse model, with both regions coexpressed in the same animal, showed significant differences that may explain how these 2 clusters are regulated differently in vivo.

Natural deletions of the region upstream of the human alpha-globin gene cluster, together with expression studies in cell lines and transgenic mice, identified a single element (HS -40) as necessary and perhaps sufficient for high-level expression of the alpha-globin genes. A similar element occupies the corresponding position upstream of the mouse (m) alpha-globin genes (mHS -26) and was thought to have similar functional properties. We knocked out mHS -26 by homologous recombination and observed the surprising result that instead of the expected severe alpha-thalassemia phenotype, the mice had a mild disease. Transcription levels of the mouse genes were reduced by about 50%, but homozygotes were healthy, with normal hemoglobin levels and only mild decreases in mean corpuscular volume and mean corpuscular hemoglobin. These results may indicate differences in the regulation of the alpha-globin clusters in mice and humans or that additional cis-acting elements remain to be characterized in one or both clusters.

To date, all of the chromosomal deletions that cause -thalassemia remove the structural  genes and/or their regulatory element (HS –40). A unique deletion occurs in a single family that juxtaposes a region that normally lies approximately 18-kilobase downstream of the human  cluster, next to a structurally normal -globin gene, and silences its expression. During development, the CpG island associated with the -globin promoter in the rearranged chromosome becomes densely methylated and insensitive to endonucleases, demonstrating that the normal chromatin structure around the -globin gene is perturbed by this mutation and that the gene is inactivated by a negative chromosomal position effect. These findings highlight the importance of the chromosomal environment in regulating globin gene expression.

We have examined the pattern of human globin gene switching in transgenic mice containing three different γ and β gene constructs (HS2GγAγδβ, HS2Aγβneo, and HS2Aγenβ) and compared the results with previously described transgenics (HS2Aγβ, HS2GγAγ-117δβ, and LCRεGγAγδβ). Developmental regulation was observed in all cases with identical patterns in lines bearing the same construct. Three different patterns of switching were observed: LCRεGγAγδβ and HS2Aγβneo mice switched rapidly, HS2GγAγδβ and HS2GγAγ-117δβ at an intermediate rate, and HS2Aγβ and HS2Aγenβ mice showed delayed switching, with a plateau in late fetal-early neonatal life and readily detectable levels of γ mRNA in adults. No difference was observed in the time of switching of the HS2GγAγδβ mice compared with those with the Aγ-117 hereditary persistence of fetal hemoglobin mutation, but adult levels of γ mRNA were significantly higher (≈5%) in lines carrying the mutation than in those without (≈1%). Reversion to the rapid switch of the LCRεGγAγδβ mice was observed in three lines with the HS2Aγβ neo construct in which expression of the tk-neo gene was approximately equal to that of the globin genes. The inclusion of the Aγ enhancer in HS2Aγβ mice did not alter the pattern of switching, or reduce the relatively high levels of γ mRNA in these lines. However, unlike other HS2 mice, the combination of HS2 and the Aγ enhancer resulted in copy number-dependent expression in HS2Aγenβ lines, with intrauterine death at ≈12.5 days gestation at high copy numbers. These results demonstrate that numerous elements throughout the β globin gene cluster interact to produce the correct pattern of developmental regulation of these genes. Furthermore, extinction of γ gene expression in adult life is not completely autonomous and is incomplete when HS2 is the only LCR element present.

Somatic cell hybrids have been used previously to investigate the developmental regulation of globin gene expression. When a chromosome containing an active human fetal gamma gene is transferred to mouse erythroleukaemia (MEL) cells by cell fusion, there is persistent expression of this gene through multiple hybrid-cell divisions despite the 'adult' trans-acting factor environment of the MEL cell. We analysed hybrids formed between embryonic erythroblasts and MEL cells to determine whether the embryonic globin genes would be similarly regulated. Surprisingly, there was no detectable expression of either the endogenous (epsilon, beta h 1) or the transgenic (human epsilon) embryonic beta-like genes in a large number of hybrid pools and clones, even though these genes were expressed in the donor cells at the time of fusion. In contrast, the human gamma gene, which is also expressed as an embryonic gene in transgenic mice, continued to be expressed in these hybrids. This lack of embryonic gene expression in hybrids also applied to the alpha gene cluster; no mouse or human zeta mRNA was detectable in embryonic cell hybrids from mice transgenic for the human alphaglobin genes. Hybrids generated from embryonic non-erythroid cells resulted in expression that was largely restricted to the adult beta gene. It has previously been suggested that epigenetic modifications to the beta-globin gene cluster during normal development are responsible for the perpetuation of gamma and beta gene expression in this hybrid cell system. The results presented here suggest that no such stable modifications occur around the embryonic genes and therefore that the regulatory mechanisms determining embryonic globin gene transcription differ from those responsible for the expression of gamma and beta genes in this system.

Somatic cell hybrids generated from transgenic mouse cells have been used to examine the developmental regulation of human gamma-to-beta-globin gene switching. In hybrids between mouse erythroleukemia (MEL) cells and transgenic erythroblasts taken at various stages of development, there was regulated expression of the human fetal gamma and adult beta genes, reproducing the in vivo pattern prior to fusion. Hybrids formed from embryonic blood cells produced predominantly gamma mRNA, whereas beta gene expression was observed in adult hybrids and a complete range of intermediate patterns was found in fetal liver hybrids. The adult environment of the MEL cells, therefore, did not appear to influence selective transcription from this gene complex. Irradiation of the embryonic erythroid cells prior to fusion resulted in hybrids containing only small fragments of donor chromosomes, but the pattern of gene expression did not differ from that of unirradiated hybrids. This finding suggests that continued expression of trans-acting factors from the donor erythroblasts is not necessary for continued expression of the human gamma gene in MEL cells. These results contrast with the lack of developmental regulation of the cluster after transfection of naked DNA into MEL cells and suggest that epigenetic processes established during normal development result in the gene cluster adopting a developmental stage-specific, stable conformation which is maintained through multiple rounds of replication and transcription in the MEL cell hybrids. On prolonged culture, hybrids that initially expressed only the gamma transgene switched to beta gene expression. The time period of switching, from approximately 10 to > 40 weeks, was similar to that seen previously in human fetal erythroblast x MEL cell hybrids but in this case bore no relationship to the time of in vivo switching. It seems unlikely, therefore, that switching in these hybrids is regulated by a developmental clock.

Expression of the human alpha and beta globin gene clusters is regulated by remote sequences, referred to as HS -40 and the beta-locus control region (beta-LCR) that lie 5-40 kb upstream of the genes they activate. Because of their common ancestry, similar organization and coordinate expression it has often been assumed that regulation of the globin gene clusters by HS -40 and the beta-LCR occurs via similar mechanisms. Using interspecific hybrids containing chromosomes with naturally occurring deletions of HS -40 we have shown that, in contrast to the beta-LCR, this element exerts no discernible effect on long-range chromatin structure and in addition does not influence formation of DNase I hypersensitive sites at the alpha globin promoters. These differences in the behaviour of HS -40 and the beta-LCR may reflect their contrasting influence on gene expression in transgenic mice and may result from the differing requirements of these elements in their radically different, natural chromosomal environments; the alpha cluster lying within a region of constitutively 'open' chromatin and the beta cluster in a segment of chromatin which opens in a tissue-specific manner. Differences in the hierarchical control of the alpha and beta globin clusters may exemplify more general differences in the regulation of eukaryotic genes which lie in similar open or closed chromosomal regions.

C16orf35 is a conserved and widely expressed gene lying adjacent to the human α-globin cluster in all vertebrate species. In-depth sequence analysis shows that C16orf35 (now called NPRL3) is an orthologue of the yeast gene Npr3 (nitrogen permease regulator 3) and, furthermore, is a paralogue of its protein partner Npr2. The yeast Npr2/3 dimeric protein complex senses amino acid starvation and appropriately adjusts cell metabolism via the TOR pathway. Here we have analysed a mouse model in which expression of Nprl3 has been abolished using homologous recombination. The predominant effect on RNA expression appears to involve genes that regulate protein synthesis and cell cycle, consistent with perturbation of the mTOR pathway. Embryos homozygous for this mutation die towards the end of gestation with a range of cardiovascular defects, including outflow tract abnormalities and ventriculoseptal defects consistent with previous observations, showing that perturbation of the mTOR pathway may affect development of the myocardium. NPRL3 is a candidate gene for harbouring mutations in individuals with developmental abnormalities of the cardiovascular system. © 2012 Springer Science+Business Media, LLC.

We have ligated two cosmids through an oligonucleotide linker to produce a single fragment spanning 70 kb of the human alpha-globin cluster, in which the alpha-like globin genes (zeta 2, alpha 2 and alpha 1), their regulatory element (HS-40) and erythroid-specific DNase I hypersensitive sites accurately retain their normal genomic organization. The zeta (embryonic) and alpha (embryonic, fetal and adult) globin genes were expressed in all 17 transgenic embryos. Similarly, all fetal and adult mice from seven transgenic lines that contained one or more copies of the fragment, produced up to 66% of the level of endogenous mouse alpha-globin mRNA. However, as for smaller constructs containing these elements, human alpha-globin expression was not copy number dependent and decreased by 1.5-9.0 fold during development. These findings suggest that either it is not possible to obtain full regulation of human alpha-globin expression in transgenic mice or, more likely, that additional alpha-globin regulatory elements lie beyond the 70 kb segment of DNA analysed.

Remote distal enhancers may be located tens or thousands of kilobases away from their promoters. How they control gene expression is still poorly understood. Here, we analyze the influence of a remote enhancer on the balance between repression (Polycomb-PcG) and activation (Trithorax-TrxG) of a developmentally regulated gene associated with a CpG island. We reveal its essential, nonredundant role in clearing the PcG complex and H3K27me3 from the CpG island. In the absence of the enhancer, the H3K27me3 demethylase (JMJD3) is not recruited to the CpG island. We propose a new role of long-range regulatory elements in removing repressive PcG complexes. © 2011 by Cold Spring Harbor Laboratory Press.

Abstract 1003 Introduction: The Congenital dyserythropoietic anemias (CDAs) are a heterogeneous group of rare inborn disorders mainly affecting erythropoiesis. Distinct from other inherited bone marrow failure syndromes, they are marked by morphological abnormalities of the erythroblasts leading to ineffective erythropoiesis/dyserythropoiesis, while the other hematopoietic lineages appear to be unaffected. Based largely on the dysplastic changes observed in erythroblasts by light and electron microscopy, the CDAs have been divided into 3 major types (CDA-1, 2 and 3). Causative genes have been identified for CDA-1 (CDAN1/15q15) and CDA-2 (CDAN2/20p11), while the chromosomal locus is known for CDA-3 (15q22). The majority of CDA-1 cases are associated with CDAN1 missense mutations, leading to single amino-acid substitutions in the 134 kDa Codanin-1 protein. Electron microscopy of CDA-1 erythroblasts reveals a characteristic pattern of spongy (“Swiss cheese”) heterochromatin. We investigated the chromatin in CDA-1 patients, the localization, distribution and interactions of Codanin-1 and generated a murine knock-out model for CDAN1. Results: Given the grossly abnormal chromatin ultrastructure in CDA-1 erythroblasts, we examined its overall content. No gross differences between normal and patient samples were seen, both in the amounts of histone proteins or various epigenetic marks of histone tails, suggesting that histone signatures, involved in maintenance of chromatin structure and epigenetic regulation, are globally maintained CDA-1. We confirmed the latter by ChIP-on-chip analysis where immunoprecipitated H4 acetylated, H3K4 dimethylated, H3K9 trimethylated and H3K27 trimethylated chromatin from CDA-1 erythroblasts and normal controls was hybridized to ENCODE genome-wide microrrays. Using a hybridoma technique, we produced 3 novel monoclonal antibodies to Codanin-1. These consistently demonstrate its distribution in both nucleus and cytoplasm of cells by immunofluorescence and Western Blotting. While Codanin-1 is found in the nucleus, it is more abundant in the cytoplasm in primary human erythroblasts. This localization pattern was unchanged in CDA-1 erythroblasts. Within the nucleus, Codanin-1 is localized in foci, while in the cytoplasm it shows an aggregated distribution, with the endoplasmic reticulum and Golgi apparatus being the most likely locations. No differences were found in the distribution patterns of RNA-polymerase-II, PML, nucleophosmin, HP1β and HP1γ between patients' and control erythroblasts. However, the localization of HP1α, a key component of heterochromatin, was found to be markedly perturbed. HP1α accumulates in the Golgi apparatus of CDA-1 but not normal erythroblasts. This was not seen in lymphoblasts derived from CDA-1 patients, nor in erythroblasts from a patient with CDA-2. We confirmed that the abnormal localization of HP1α in CDA-1 patients is confined to the intermediate erythroblast maturation stage, where the characteristic ultrastructural chromatin pattern of CDA-1 is observed. Immunoprecipitation of erythroblast protein extracts with anti- HP1α antibodies co-precipitated Codanin-1, suggesting that an abnormality in Codanin-1 could be responsible for the aberrant localization of HP1α. By confocal immunofluorescence, we also found Codanin-1 to co-localize partially with SEC23B, the protein mutated in CDA-2, suggesting a molecular link between the two major types of CDA. We generated the first murine Cdan1 gene-trap model demonstrating its widespread expression during embryonic development. Cdan1gt/gt homozygotes die in-utero before the onset of primitive erythropoiesis, suggesting that Cdan1 has other critical roles during embryogenesis. Adult heterozygous Cdan1wt/gt mice had normal hematopoietic function and morphology, normal life expectancy and fertility. No extra-hematopoietic macroscopic phenotype could be identified. Conclusions: Our results show that the absence of the highly conserved and ubiquitously expressed Codanin-1 is embryonic lethal, and suggest that missense CDAN1 mutations are responsible for the erythroid specific phenotype of CDA-1 via the abnormal cellular trafficking of the heterochromatin protein HP1α. Moreover, our findings suggest that common molecular pathways underlie the CDAs. Disclosures: No relevant conflicts of interest to declare.

The beta-globin gene complex is regulated by an upstream locus control region (LCR) which is responsible for high-level, position-independent, erythroid-cell-specific expression of the genes in the cluster. Its role in the developmental regulation of beta-like globin gene transcription remains to be established. We have examined the effect of a single LCR element, hypersensitive site 2 (HS2), on the developmental regulation of the human fetal gamma and adult beta genes in transgenic mice. In mice bearing HS2A gamma beta and HS2G gamma A gamma-117 delta beta human globin gene constructs, switching from gamma- to beta-gene expression begins at about day 13.5 of gestation and is largely completed shortly after birth. The larger construct also demonstrates a switch in G gamma- to A gamma-gene expression during the gamma-to-beta switch similar to that observed during normal human development. We conclude that HS2 alone is sufficient for developmental regulation of the human beta-globin genes.

Previous studies in the mouse have shown that high levels of α-globin gene expression in late erythropoiesis depend on long-range, physical interactions between remote upstream regulatory elements and the globin promoters. Using quantitative chromosome conformation capture (q3C), we have now analyzed all interactions between 4 such elements lying 10 to 50 kb upstream of the human α cluster and their interactions with the α-globin promoter. All of these elements interact with the α-globin gene in an erythroid-specific manner. These results were confirmed in a mouse model of human α globin expression in which the human cluster replaces the mouse cluster in situ (humanized mouse). We have also shown that expression and all of the long-range interactions depend largely on just one of these elements; removal of the previously characterized major regulatory element (called HS −40) results in loss of all the interactions and α-globin expression. Reinsertion of this element at an ectopic location restores both expression and the intralocus interactions. In contrast to other more complex systems involving multiple upstream elements and promoters, analysis of the human α-globin cluster during erythropoiesis provides a simple and tractable model to understand the mechanisms underlying long-range gene regulation.

The major positive regulatory activity of the human alpha-globin gene complex has been localized to an element associated with a strong erythroid-specific DNase I hypersensitive site (HS -40) located 40 kb upstream of the zeta 2-globin mRNA cap site. Footprint and gel shift analyses of the element have demonstrated the presence of four binding sites for the nuclear factor GATA-1 and two sites corresponding to the AP-1 consensus binding sequence. This region resembles one of the major elements of the beta-globin locus control region in its constitution and characteristics; this together with evidence from expression studies suggests that HS -40 is a primary element controlling alpha-globin gene expression.

The analysis of globin gene regulation has elucidated many of the principles underlying mammalian gene expression. To understand how the epigenetic program that unfolds during hematopoiesis impacts on alpha (α)-globin gene expression, we analyzed DNA methylation at over 200 CpG dinucleotides in 130 kb around the human α-globin gene cluster focussing particularly on a region (12 kb) covering the α2 and α1-globin genes (HBA2 and HBA1), and the upstream regulatory sites (DNAse I hypersensitive sites (HS) -48, HS-40, HS-33, and HS-10); so-called multi-species conserved sequencesregulatory element1-4 (MCS-R1-4). Cultures of primary adult erythroid cells (at three stages: early, intermediate, and late) were analyzed together with non-erythroid human cells (neutrophils, peripheral blood mononuclear cells, and a human embryonic stem (ES) cell line). Using a recently published method involving bisulphite modification and MALDI-TOF Mass Spectrometry we quantified the level of DNA methylation across the alpha globin cluster. Most sequences outside of the CpG islands were methylated. However, the DNA methylation levels between erythroid and non-erythroid cells differed at regulatory elements. MCS-R1 contained only 1 CpG site and the average percentage of DNA methylation for erythroid and non-erythroid was 8% and 40%, respectively. While they were 8% vs. 42% (p=0.428), 11% vs. 99% (p=0.054), and 14% vs. 97% (p=0.001) in MCS-R2 to 4, respectively. To study changes in DNA methylation in a comprehensive, developmental and tissue-specific manner, we used a humanized-mouse model, in which the conserved α-globin syntenic region of mouse genomic sequence is replaced by that of human. This contains all cis-acting sequences required for fully regulated expression of the α-globin genes. Although the alpha globin cluster is expressed efficiently in this model, DNA methylation patterns differed in the 20 kb region encompassing the α-globin genes when comparing primary human cells and primary humanized cells. DNA in humanized cells were relatively hypomethylated both in erythroid and non-erythroid tissues. The methylation levels were higher in testis of the humanized mouse than in other tissues but still not as high as those in normal human non-erythroid cells. The cause of these differences is being investigated. Despite these differences, the humanized-mouse mimicked the patterns of methylation found in regulatory elements in erythroid and nonerythroid cells. This study shows that despite appropriate regulation of gene expression, epigenetic templating may differ between species (human and mouse). DNA methylation at the upstream regulatory elements might be involved in the regulation of α-globin gene expression during erythropoiesis, although it is equally possible that these changes in methylation are generated by passive demethylation secondary to transcription factor binding in erythroid cells. The mechanism(s) underlying demethylation of regulatory elements during differentiation remains to be clarified.

Extreme skewing of X-chromosome inactivation (XCI) is rare in the normal female population but is observed frequently in carriers of some X-linked mutations. Recently, it has been shown that various forms of X-linked mental retardation (XLMR) have a strong association with skewed XCI in female carriers, but the mechanisms underlying this skewing are unknown. ATR-X syndrome, caused by mutations in a ubiquitously expressed, chromatin-associated protein, provides a clear example of XLMR in which phenotypically normal female carriers virtually all have highly skewed XCI biased against the X chromosome that harbors the mutant allele. Here, we have used a mouse model to understand the processes causing skewed XCI. In female mice heterozygous for a null Atrx allele, we found that XCI is balanced early in embryogenesis but becomes skewed over the course of development, because of selection favoring cells expressing the wild-type Atrx allele. Unexpectedly, selection does not appear to be the result of general cellular-viability defects in Atrx-deficient cells, since it is restricted to specific stages of development and is not ongoing throughout the life of the animal. Instead, there is evidence that selection results from independent tissue-specific effects. This illustrates an important mechanism by which skewed XCI may occur in carriers of XLMR and provides insight into the normal role of ATRX in regulating cell fate.

We have investigated the transcriptional regulation of the human embryonic zeta-globin gene promoter. First, we examined the effect that deletion of sequences 5' to zeta-globin's CCAAT box have on zeta-promoter activity in erythroid cell lines. Deletions of sequences between -116 and -556 (cap = 0) had little effect while further deletion to -84 reduced zeta-promoter activity by only 2-3-fold in both transiently and stably transfected erythroid cells. Constructs containing 67, 84 and 556 bp of zeta-globin 5' flanking region linked to a beta-galactosidase reporter gene (lacZ) and hypersensitive site -40 (HS-40) of the human alpha-globin gene cluster were then employed for the generation of transgenic mice. LacZ expression from all constructs, including a 67 bp zeta-globin promoter, was erythroid-specific and most active between 8.5 and 10.5 days post-fertilisation. By 16.5 days gestation, lacZ expression dropped 40-100-fold. These results suggest that embryonic-specific activation of the human zeta-globin promoter is conferred by a 67 bp zeta-promoter fragment containing only a CCAAT and TATA box.

A 350-bp segment of DNA associated with an erythroid-specific DNase I-hypersensitive site (HS-40), upstream of the alpha-globin gene cluster, has been identified as the major tissue-specific regulator of the alpha-globin genes. However, this element does not direct copy number-dependent or developmentally stable expression of the human genes in transgenic mice. To determine whether additional upstream hypersensitive sites could provide more complete regulation of alpha gene expression we have studied 17 lines of transgenic mice bearing various DNA fragments containing HSs -33, -10, -8, and -4, in addition to HS -40. Position-independent, high-level expression of the human zeta- and alpha-globin genes was consistently observed in embryonic erythroid cells. However, the additional HSs did not confer copy-number dependence, alter the level of expression, or prevent the variable down-regulation of expression in adults. These results suggest that the region upstream of the human alpha-globin genes is not equivalent to that upstream of the beta locus and that although the two clusters are coordinately expressed, there may be differences in their regulation.

Abstract 4060 Poster Board III-995 Expression of many tissue and developmental stage specific genes is controlled by remote regulatory elements. The mammalian a-globin gene cluster includes four previously characterised remote regulatory elements (MCS-R1 to R4) upstream of the a-globin genes. Naturally occurring deletions and experiments in transgenic mice, have shown that one or more of these elements are necessary for fully regulated expression of the a-like globin genes. At present it is unclear exactly how these elements interact with the a-globin genes to enhance their expression, although ultimately we know that they physically interact with the promoters forming a chromatin loop. Of interest, three of the MCS-R sequences are located within introns of a widely expressed gene C16orf35, which lies adjacent to the a-globin locus and we now know that such complex, intermingled arrangements are common in the mammalian genome. We have asked two questions: can the MCS-R elements be transcribed while activating the globin genes, and does transcription of the MCS-R elements play any role in their activation? We have analysed nascent transcription of C16orf35 and a-globin, and shown that both genes are simultaneously transcribed in a high proportion of erythroid cells. Thus it appears that the MCS-R elements can interact with the globin genes while being transcribed. We next asked whether transcription of the C16orf35 gene (through the upstream regulatory elements) is necessary for their activation. The promoter region and a putative erythroid-specific promoter of the C16orf35 gene were deleted (singly and in combination) by homologous recombination in mice. Provisional analysis has shown no evidence of a-thalassaemia in these mice, suggesting that activation of the MCS-R elements occurs independently of their transcription. In addition to their importance in globin gene regulation, these findings have general implications for the relationship between structure and function in the mammalian genome and the mechanisms by which long-range elements interact with their cognate promoters. Disclosures: No relevant conflicts of interest to declare.

We have identified a remote, tissue-specific, positive regulatory element that is of major importance in determining the level of human alpha-globin gene expression. Stable transformants containing this DNA segment linked to the alpha gene in mouse erythroleukemia cells expressed human alpha mRNA at levels that are indistinguishable from those seen in interspecific hybrids containing the human alpha genes in their normal context on chromosome 16. Furthermore, all transgenic mice containing the alpha genes linked to this region expressed alpha-globin mRNA at high levels in erythroid tissues; and in one such mouse, readily detectable levels of human alpha-globin chains could be demonstrated in the peripheral blood. There is considerable similarity in the position, structure, and function of this region upstream of the alpha-globin complex with previously described elements within the beta-globin dominant control region (DCR). This is m marked contrast to other structural and functional differences between the two gene clusters. It seems likely that these critical, positive regulatory regions might provide target sequences through which coordinate regulation of the alpha- and beta-like globin genes is achieved.

We have identified and cloned the major alpha globin locus regulatory element in the mouse (m alpha RE). This element shows a high level of sequence homology to its human counterpart (HS -40) and lies between the same two exons of an upstream, widely expressed gene in both species. Footprinting and band shift studies of the core element show conservation of many (but not all) of the protein binding sites identified as functionally important in HS -40. The functional equivalence of the mouse element was shown by attaching it to a human alpha globin gene and examining expression in transgenic mice. Readily detectable levels of human alpha mRNA were produced in these mice but they were lower than the endogenous gene expression and did not show copy number dependence. These results suggest that sequences additional to this major regulatory element may be necessary to obtain complete regulation of the alpha globin genes in both species.