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Author(s): Elizabeth A Traxler [1, 2]; Yu Yao [1]; Yong-Dong Wang [3]; Kaitly J Woodard [1]; Ryo Kurita [4]; Yukio Nakamura [4, 5]; Jim R Hughes [6]; Ross C Hardison [...]

Germline GATA1 mutations that result in the production of an amino-truncated protein termed GATA1s (where s indicates short) cause congenital hypoplastic anemia. In patients with trisomy 21, similar somatic GATA1s-producing mutations promote transient myeloproliferative disease and acute megakaryoblastic leukemia. Here, we demonstrate that induced pluripotent stem cells (iPSCs) from patients with GATA1-truncating mutations exhibit impaired erythroid potential, but enhanced megakaryopoiesis and myelopoiesis, recapitulating the major phenotypes of the associated diseases. Similarly, in developmentally arrested GATA1-deficient murine megakaryocyte-erythroid progenitors derived from murine embryonic stem cells (ESCs), expression of GATA1s promoted megakaryopoiesis, but not erythropoiesis. Transcriptome analysis revealed a selective deficiency in the ability of GATA1s to activate erythroid-specific genes within populations of hematopoietic progenitors. Although its DNA-binding domain was intact, chromatin immunoprecipitation studies showed that GATA1s binding at specific erythroid regulatory regions was impaired, while binding at many nonerythroid sites, including megakaryocytic and myeloid target genes, was normal. Together, these observations indicate that lineage-specific GATA1 cofactor associations are essential for normal chromatin occupancy and provide mechanistic insights into how GATAIs mutations cause human disease. More broadly, our studies underscore the value of ESCs and iPSCs to recapitulate and study disease phenotypes., Introduction GATA1 is an essential erythro-megakaryocytic transcription factor. In normal humans, alternative splicing or translation initiation site usage produces 2 forms of GATA1 protein: full-length (GATA1fl) and a truncated isoform [...]

The laboratory mouse shares the majority of its protein-coding genes with humans, making it the premier model organism in biomedical research, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications and replication domains throughout the mouse genome in diverse cell and tissue types. By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases., Author(s): Feng Yue [1, 2]; Yong Cheng [3]; Alessandra Breschi [4]; Jeff Vierstra [5]; Weisheng Wu [6]; Tyrone Ryba [7]; Richard Sandstrom [5]; Zhihai Ma [3]; Carrie Davis [8]; Benjamin [...]

Recent advances in genomic research have revealed the existence of a large number of transcripts devoid of protein-coding potential in multiple organisms (1-8). Although the functional role for long non-coding [...]

Hematopoietic stem and progenitor cell (HSPC) expansion is regulated by intrinsic signaling pathways activated by cytokines. The intracellular kinase JAK2 plays an essential role in cytokine signaling, and activating mutations in JAK2 are found in a number of hematologic malignancies. We previously demonstrated that lymphocyte adaptor protein (Lnk, also known as Sh2b3) binds JAK2 and attenuates its activity, thereby limiting HSPC expansion. Here we show that loss of Lnk accelerates and exacerbates oncogenic JAK2-induced myeloproliferative diseases (MPDs) in mice. Specifically, Lnk deficiency enhanced cytokine-independent JAK/STAT signaling and augmented the ability of oncogenic JAK2 to expand myeloid progenitors in vitro and in vivo. An activated form of JAK2, unable to bind Lnk, caused greater myeloid expansion than activated JAK2 alone and accelerated myelofibrosis, indicating that Lnk directly inhibits oncogenic JAK2 in constraining MPD development. In addition, Lnk deficiency cooperated with the BCR/ABL oncogene, the product of which does not directly interact with or depend on JAK2 or Lnk, in chronic myeloid leukemia (CML) development, suggesting that Lnk also acts through endogenous pathways to constrain HSPCs. Consistent with this idea, aged [Lnk.sup.[-/-]] mice spontaneously developed a CML-like MPD. Taken together, our data establish Lnk as a bona fide suppressor of MPD in mice and raise the possibility that Lnk dysfunction contributes to the development of hematologic malignancies in humans., Introduction JAK2 plays an essential role in the signaling of receptors for many cytokines, which include thrombopoietin (Tpo), erythropoietin (Epo), and granulocyte-CSF (G-CSF) (1). JAK2-deficient mice die of anemia at [...]

The transcription factor GATA-1 and its cofactor, friend of GATA-1 (FOG-1), are essential for normal erythroid development. FOG-1 physically interacts with GATA-1 to augment or inhibit its activity. The mechanisms by which FOG-1 regulates GATA-1 function are unknown. By using an assay that is based on the phenotypic rescue of a GATA-1-null erythroid cell line, we found that a conditional form of GATA-1 (GATA-1-ER) strongly induced histone acetylation at the [beta]-major globin promoter in vivo, consistent with previous results. In contrast, GATA-1 bearing a point mutation that impairs FOG-1 binding [GATA-1(V205M)-ER] failed to induce high levels of histone acetylation at this site. However, at DNase l-hypersensitive site (HS)3 of the [beta]-globin locus control region, GATA-1-induced histone acetylation was FOG-1-independent. Because the V205M mutation does not disrupt GATA-1 binding to DNA templates in vitro, we were surprised to find that in vivo GATA-1(V205M)-ER fails to bind the [beta]-globin promoter. However, at HS3, DNA binding by GATA-1 was FOG-1-independent, thus correlating histone acetylation with GATA-1 occupancy. Examination of additional GATA1-dependent regulatory elements showed that the interaction with FOG-1 is required for GATA-1 occupancy at select sites, such as HS2, but is dispensable at others, including the FOG-1-independent GATA-1 target gene EKLF. Remarkably, at the GATA-2 gene, which is repressed by GATA-1, interaction with FOG-1 was dispensable for GATA-1 occupancy and was required for transcriptional inhibition and histone deacetylation. These results indicate that FOG-1 employs distinct mechanisms when cooperating with GATA-1 during transcriptional activation and repression. globin | chromatin | histones | acetylation

The hematopoietic transcription factor GATA-1 regulates erythropoiesis And [beta]-globin expression. Although consensus GATA-1 binding sites exist throughout the murine [beta]-globin locus, we found that GATA-1 discriminates among these sites in vivo. Conditional expression of GATA-1 in GATA-1-null cells recapitulated the occupancy pattern. GATA-1 induced RNA polymerase II (pol II) recruitment to subregions of the locus control region and to the [beta]-globin promoters. The hematopoietic factor NF-E2 cooperated with GATA-1 to recruit pol II to the promoters. We propose that only when GATA-1 attracts pol II to the locus control region can pol II access the promoter in a NF-E2-dependent manner.

Author(s): Anthony J. Kihm [1]; Yi Kong [1]; Wei Hong [1]; J. Eric Russell [1]; Susan Rouda [1]; Kazuhiko Adachi [1]; M. Celeste Simon [2]; Gerd A. Blobel [1]; Mitchell [...]

The transcription factor GATA-1 coordinates multiple events during terminal erythroid cell maturation. GATA-1 participates in the transcription of virtually all erythroid-specific genes, blocks apoptosis of precursor cells, and controls the balance between proliferation and cell cycle arrest. Prior studies suggest that the function of GATA-1 is mediated in part through association with transcriptional cofactors. CREB-binding protein (CBP) and its close relative p300 serve as coactivators for a variety of transcription factors involved in growth control and differentiation. We report here that CBP markedly stimulates GATA-1's transcriptional activity in transient transfection experiments in nonhematopoietic cells. GATA-1 and CBP also coimmunoprecipitate from nuclear extracts of erythroid cells. Interaction mapping pinpoints contact sites to the zinc finger region of GATA-I and to the E1A-binding region of CBP. Expression of a conditional form of adenovirus E1A in murine erythroleukemia cells blocks differentiation and expression of endogenous GATA-1 target genes, whereas mutant forms of E1A unable to bind CBP/p300 have no effect. Our findings add GATA-1, and very likely other members of the GATA family, to the growing list of molecules implicated in the complex regulatory network surrounding CBP/p300.