scholarly journals Detection in non-erythroid cells of a factor with the binding characteristics of the erythroid cell transcription factor EF1

1990 ◽  
Vol 269 (2) ◽  
pp. 543-545
Author(s):  
N D Perkins ◽  
K H Orchard ◽  
M L K Collins ◽  
D S Latchman ◽  
G H Goodwin
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Critical Role ◽  
Cell Types ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
First Report ◽  
Binding Characteristics

The erythroid transcription factor erythroid factor-1 (EF1) plays a critical role in the transcription of erythroid-specific genes. Here we report the presence of a factor with the mobility and sequence-specific DNA-binding characteristics of EF1 at low abundance in a wide variety of non-erythroid cell types. This is the first report of an EF1-like activity in non-erythroid cells and indicates that this factor may play a role in the regulation of genes expressed in such cells.

Positive regulators of the lineage-specific transcription factor GATA-1 in differentiating erythroid cells

1994 ◽  
Vol 14 (5) ◽  
pp. 3108-3114
Author(s):  
M H Baron ◽  
S M Farrington
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Transcription Factor ◽  
Embryonic Stem Cells ◽  
Cultured Cells ◽  
Globin Gene ◽  
Embryonic Stem ◽  
Targeted Mutagenesis ◽  
Erythroid Cell ◽  
Erythroid Cells

The zinc finger transcription factor GATA-1 is a major regulator of gene expression in erythroid, megakaryocyte, and mast cell lineages. GATA-1 binds to WGATAR consensus motifs in the regulatory regions of virtually all erythroid cell-specific genes. Analyses with cultured cells and cell-free systems have provided strong evidence that GATA-1 is involved in control of globin gene expression during erythroid differentiation. Targeted mutagenesis of the GATA-1 gene in embryonic stem cells has demonstrated its requirement in normal erythroid development. Efficient rescue of the defect requires an intact GATA element in the distal promoter, suggesting autoregulatory control of GATA-1 transcription. To examine whether GATA-1 expression involves additional regulatory factors or is maintained entirely by an autoregulatory loop, we have used a transient heterokaryon system to test the ability of erythroid factors to activate the GATA-1 gene in nonerythroid nuclei. We show here that proerythroblasts and mature erythroid cells contain a diffusible activity (TAG) capable of transcriptional activation of GATA-1 and that this activity decreases during the terminal differentiation of erythroid cells. Nuclei from GATA-1- mutant embryonic stem cells can still be reprogrammed to express their globin genes in erythroid heterokaryons, indicating that de novo induction of GATA-1 is not required for globin gene activation following cell fusion.

The cellular transcription factor CREB corresponds to activating transcription factor 47 (ATF-47) and forms complexes with a group of polypeptides related to ATF-43

1990 ◽  
Vol 10 (12) ◽  
pp. 6192-6203
Author(s):  
H C Hurst ◽  
N Masson ◽  
N C Jones ◽  
K A Lee
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Multigene Family ◽  
Protein Complexes ◽  
Heat Stability ◽  
Cell Types ◽  
Transcriptional Level ◽  
Sequence Motif ◽  
Cellular Transcription Factor ◽  
Activating Transcription Factor

Promoter elements containing the sequence motif CGTCA are important for a variety of inducible responses at the transcriptional level. Multiple cellular factors specifically bind to these elements and are encoded by a multigene family. Among these factors, polypeptides termed activating transcription factor 43 (ATF-43) and ATF-47 have been purified from HeLa cells and a factor referred to as cyclic AMP response element-binding protein (CREB) has been isolated from PC12 cells and rat brain. We demonstrated that CREB and ATF-47 are identical and that CREB and ATF-43 form protein-protein complexes. We also found that the cis requirements for stable DNA binding by ATF-43 and CREB are different. Using antibodies to ATF-43 we have identified a group of polypeptides (ATF-43) in the size range from 40 to 43 kDa. ATF-43 polypeptides are related by their reactivity with anti-ATF-43, DNA-binding specificity, complex formation with CREB, heat stability, and phosphorylation by protein kinase A. Certain cell types vary in their ATF-43 complement, suggesting that CREB activity is modulated in a cell-type-specific manner through interaction with ATF-43. ATF-43 polypeptides do not appear simply to correspond to the gene products of the ATF multigene family, suggesting that the size of the ATF family at the protein level is even larger than predicted from cDNA-cloning studies.

scholarly journals Direct effects of thyroid hormones on bone marrow erythroid cells of rats

Blood ◽  
1975 ◽  
Vol 45 (5) ◽  
pp. 671-679 ◽  
Author(s):  
LA Malgor ◽  
CC Blanc ◽  
E Klainer ◽  
SE Irizar ◽  
PR Torales ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Thyroid Hormones ◽  
Rabbit Antiserum ◽  
Cell Types ◽  
Progressive Increase ◽  
Erythroid Cell ◽  
Control Group ◽  
Erythroid Cells ◽  
Intravenous Infusions ◽  
Marrow Cellularity

Abstract A stimulatory effect on bone marrow cellularity was observed in normal and nephrectomized rats continuously infused with T3 and T4. Results of bone marrow studies are expressed in absolute numbers of total nucleated erythroid cells per milligram of femoral marrow at the beginning and after 8 hr of continuous intravenous infusions. Administration of T3 and T4 to nephrectomized rats produced a marked and significant increase in total erythroid cells counted. After differential analyses of the nucleated erythroid elements, a significant increase in all erythroid cell types was also observed. Similar results were seen in a control group of rats in which both ureters have been previously ligated and in groups of nephrectomized rats receiving rabbit antiserum against erythropoietin before starting the intravenous infusions of T3 and T4. These results indicate that stimulation of marrow erythropoiesis produced by thyroid hormones in our system is not dependent on renal or extra-renal production of erythropoietin. The progressive introduction of T3 and T4 into the circulation of rats with bilateral nephrectomy or ureter-ligated normal rats, may overload the mechanism of transport of these hormones in plasma. As a consequence, a progressive increase in free active forms of T3 and T4 in plasma may occur. Our interpretation of the present findings is that thyroid hormones stimulate directly bone marrow erythropoiesis. This stimulation is clearly evident when high levels of free active forms of thyroid hormones are present in plasma.

scholarly journals Coordination of erythropoiesis by the transcription factor c-Myb

Blood ◽  
2006 ◽  
Vol 107 (12) ◽  
pp. 4703-4710 ◽  
Author(s):  
Alexandros Vegiopoulos ◽  
Paloma García ◽  
Nikla Emambokus ◽  
Jon Frampton
Keyword(s):  
Transcription Factor ◽  
Critical Role ◽  
Leukemia Cell ◽  
Erythroid Cell ◽  
Erythroid Progenitors ◽  
Aberrant Expression ◽  
Protein Levels ◽  
Kit Receptor ◽  
Erythroid Cell Differentiation ◽  
Factor C

Abstract The involvement of the transcription factor c-Myb in promoting the proliferation and inhibition of erythroid cell differentiation has been established in leukemia cell models. The anemia phenotype observed in c-myb knockout and knockdown mice highlights a critical role for c-Myb in erythropoiesis. However, determining the reason for the failure of erythropoiesis in these mice and the precise function of c-Myb in erythroid progenitors remains elusive. We examined erythroid development under conditions of reduced c-Myb protein levels and report an unexpected role for c-Myb in the promotion of commitment to the erythroid lineage and progression to erythroblast stages. c-myb knockdown erythroid colony-forming unit (CFU-E) stage progenitors displayed an immature phenotype and aberrant expression of several hematopoietic regulators. To extend our findings, we analyzed the response of normal enriched erythroid progenitors to inducible disruption of a floxed c-myb allele. In agreement with the c-myb knockdown phenotype, we show that c-Myb is strictly required for expression of the c-Kit receptor in erythroid cells.

Heme-Regulated Inhibitor (HRI) Activates Transcription Factor ATF4 to Promote BCL11A Transcription and Fetal Hemoglobin Silencing

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 814-814
Author(s):  
Peng Huang ◽  
Scott A. Peslak ◽  
Xianjiang Lan ◽  
Eugene Khandros ◽  
Malini Sharma ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cell Line ◽  
Research Funding ◽  
Fetal Hemoglobin ◽  
Erythroid Cell ◽  
Mrna Levels ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Major Pathway ◽  
Specific Regulation

Reactivation of fetal hemoglobin in adult red blood cells benefits patients with sickle cell disease and β-thalassemia. BCL11A is one of the predominant repressors of fetal γ-globin transcription and stands as an appealing target for therapeutic genome manipulation. However, pharmacologic perturbation of BCL11A function or its co-regulators remains an unmet challenge. Previously, we reported the discovery of the erythroid-enriched protein kinase HRI as a novel regulator of γ-globin transcription and found that HRI functions in large part via controlling the levels of BCL11A transcription (Grevet et al., Science, 2018). However, the specific mechanisms underlying HRI-mediated modulation of BCL11A levels remain unknown. To identify potential HRI-controlled transcription factors that regulate BCL11A, we performed a domain-focused CRISPR screen that targeted the DNA binding domains of 1,447 genes in the human erythroid cell line HUDEP2. Activating transcription factor 4 (ATF4) emerged as a novel γ-globin repressor. Prior studies reported that ATF4 production is under positive influence of HRI. Specifically, HRI phosphorylates translation factor EIF2α which in turn augments translation of ATF4 mRNA. As expected, HRI deficiency reduced ATF4 protein amounts in HUDEP2 and primary erythroid cells. We further found that the degree of γ-globin reactivation was similar in ATF4 and HRI-depleted cells. ATF4 ChIP-seq in both HUDEP2 and primary erythroblast identified 4,547 and 3,614 high confidence binding sites, respectively. Notably, we did not observe significant enrichment of ATF4 binding or even the presence of an ATF4 consensus motif at the γ-globin promoters, suggesting that ATF4 regulates the γ-globin genes indirectly. However, ATF4 specifically bound to one of the three major BCL11A erythroid enhancers (+55) in both cell types. This was the sole binding site within the ~0.5Mb topologically associating domain that contains the BCL11A gene. Eliminating this ATF4 motif via CRISPR guided genome editing lowered BCL11A mRNA levels and increased γ-globin transcription. Capture-C showed that ATF4 knock-out or removal of the ATF4 site at the BCL11A (+55) enhancer decreased chromatin contacts with the BCL11A promoter. Forced expression of BCL11A largely restored γ-globin silencing in cells deficient for ATF4 or lacking the ATF4 motif in the BCL11A (+55) enhancer. An unexplained observation from our prior study was that HRI loss did not significantly lower Bcl11a levels in murine erythroid cells. Therefore, we mutated the analogous ATF4 motif in the Bcl11a enhancer in the murine erythroid cell line G1E. Unlike in human cells, Bcl11a mRNA synthesis was decreased only very modestly, and there was no effect on the murine embryonic globin genes whose silencing requires Bcl11a. This suggests that the species specific regulation of BCL11A by HRI results from divergent functional roles of ATF4 binding at the BCL11A (+55) enhancer. In sum, our studies uncover a major pathway that extends linearly from HRI to ATF4 to BCL11A to γ-globin. Moreover, these results further support HRI as a pharmacologic target for the selective regulation of BCL11A and γ-globin. Disclosures Blobel: Pfizer: Research Funding; Bioverativ: Research Funding.

scholarly journals AP1 Regulation of Proliferation and Initiation of Apoptosis in Erythropoietin-Dependent Erythroid Cells

1998 ◽  
Vol 18 (7) ◽  
pp. 3699-3707 ◽  
Author(s):  
Sarah M. Jacobs-Helber ◽  
Amittha Wickrema ◽  
Michael J. Birrer ◽  
Stephen T. Sawyer
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Dominant Negative ◽  
Stress Factors ◽  
Erythroid Cell ◽  
Mrna Levels ◽  
Erythroid Cells ◽  
Dna Binding Activity ◽  
Regulation Of Proliferation ◽  
Induction Of Apoptosis

ABSTRACT The transcription factor AP1 has been implicated in the induction of apoptosis in cells in response to stress factors and growth factor withdrawal. We report here that AP1 is necessary for the induction of apoptosis following hormone withdrawal in the erythropoietin (EPO)-dependent erythroid cell line HCD57. AP1 DNA binding activity increased upon withdrawal of HCD57 cells from EPO. A dominant negative AP1 mutant rendered these cells resistant to apoptosis induced by EPO withdrawal and blocked the downregulation of Bcl-XL. JunB is a major binding protein in the AP1 complex observed upon EPO withdrawal; JunB but not c-Jun was present in the AP1 complex 3 h after EPO withdrawal in HCD57 cells, with a concurrent increase injunB message and protein. Furthermore, analysis of AP1 DNA binding activity in an apoptosis-resistant subclone of HCD57 revealed a lack of induction in AP1 DNA binding activity and no change injunB mRNA levels upon EPO withdrawal. In addition, we determined that c-Jun and AP1 activities correlated with EPO-induced proliferation and/or protection from apoptosis. AP1 DNA binding activity increased over the first 3 h following EPO stimulation of HCD57 cells, and suppression of AP1 activity partially inhibited EPO-induced proliferation. c-Jun but not JunB was present in the AP1 complex 3 h after EPO addition. These results implicate AP1 in the regulation of proliferation and survival of erythroid cells and suggest that different AP1 factors may play distinct roles in both triggering apoptosis (JunB) and protecting erythroid cells from apoptosis (c-Jun).

The formation and maintenance of the definitive endoderm lineage in the mouse: involvement of HNF3/forkhead proteins

Development ◽  
1993 ◽  
Vol 119 (4) ◽  
pp. 1301-1315 ◽  
Author(s):  
S.L. Ang ◽  
A. Wierda ◽  
D. Wong ◽  
K.A. Stevens ◽  
S. Cascio ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Neural Tube ◽  
Critical Role ◽  
Primitive Streak ◽  
Cell Types ◽  
Definitive Endoderm ◽  
Gut Development ◽  
Binding Assays ◽  
Anterior Portion ◽  
Endoderm Development

Little is known about genes that govern the development of the definitive endoderm in mammals; this germ layer gives rise to the intestinal epithelium and various other cell types, such as hepatocytes, derived from the gut. The discovery that the rat hepatocyte transcription factor HNF3 is similar to the Drosophila forkhead gene, which plays a critical role in gut development in the fly, led us to isolate genes containing the HNF3/forkhead (HFH) domain that are expressed in mouse endoderm development. We recovered mouse HNF3 beta from an embryo cDNA library and found that the gene is first expressed in the anterior portion of the primitive streak at the onset of gastrulation, in a region where definitive endoderm first arises. Its expression persists in axial structures derived from the mouse equivalent of Hensen's node, namely definitive endoderm and notochord, and in the ventral region of the developing neural tube. Expression of the highly related gene, HNF3 alpha, appears to initiate later than HNF3 beta and is first seen in midline endoderm cells. Expression subsequently appears in notochord, ventral neural tube, and gut endoderm in patterns similar to HNF3 beta. Microscale DNA binding assays show that HNF3 proteins are detectable in the midgut at 9.5 days p.c. At later stages HNF3 mRNAs and protein are expressed strongly in endoderm-derived tissues such as the liver. HNF3 is also the only known hepatocyte-enriched transcription factor present in a highly de-differentiated liver cell line that retains the capacity to redifferentiate to the hepatic phenotype. Taken together, these studies suggest that HNF3 alpha and HNF3 beta are involved in both the initiation and maintenance of the endodermal lineage. We also discovered a novel HFH-containing gene, HFH-E5.1, that is expressed transiently in posterior ectoderm and mesoderm at the primitive streak stage, and later predominantly in the neural tube. HFH-E5.1 is highly similar in structure and expression profile to the Drosophila HFH gene FD4, suggesting that HFH family members have different, evolutionarily conserved roles in development.

Granule cell specification in the developing mouse brain as defined by expression of the zinc finger transcription factor RU49

Development ◽  
1996 ◽  
Vol 122 (2) ◽  
pp. 555-566 ◽  
Author(s):  
X.W. Yang ◽  
R. Zhong ◽  
N. Heintz
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Granule Cell ◽  
Ventricular Zone ◽  
Critical Role ◽  
Neuronal Cell ◽  
Developing Brain ◽  
Granule Neurons ◽  
Cell Specification ◽  
Rhombic Lip

The creation of specific neuronal cell types within the developing brain is a critical and unsolved biological problem. Precedent from invertebrate development, and from vertebrate myogenesis and lymphogenesis, has established that cell specification often involves transcription factors that are expressed throughout the differentiation of a given cell type. In this study, we have identified in Zn2+ finger transcription factor RU49 as a definitive marker for the cerebellar granule neuron lineage. Thus, RU49 is expressed in the earliest granule cell progenitors at the rhombic lip as they separate from the ventricular zone of the neural tube to generate a secondary proliferative matrix, and it continues to be expressed in differentiating and mature granule neurons. Proliferating granule cell progenitors isolated from the rhombic lip at E14 or from the external germinal layer at P6 continue to express RU49 in vitro. Both the olfactory bulb and dentate gyrus granule cell lineages also express this factor as they are generated with the developing brain. RU49 binds a novel bipartite DNA-binding element in a manner consistent with chemical rules governing the DNA-binding specificity of this class of transcription factor. The novel biochemical properties of RU49 and its restricted expression within the three lineages of CNS granule neurons suggest that RU49 may play a critical role in their specification. Furthermore, these results raise the interesting possibility that the generation of these three neuronal populations to form displaced germinative zones within the developing brain may reflect their use of a common developmental mechanism involving RU49.

Role of Atypical p50:p50:Bcl-3 NFκlB Complex in Differentiation of Erythroid Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 568-568 ◽  
Author(s):  
Stephen Sawyer ◽  
Jingchun Chen
Keyword(s):  
Dna Binding ◽  
Transcriptional Control ◽  
Biological Significance ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Necrosis Factor Alpha ◽  
Erythroid Progenitor ◽  
Tnf Α

Abstract We recently reported that mouse and human primary erythroid progenitor cells and erythroid cell lines synthesize and respond to Tumor Necrosis Factor-alpha (TNF-α). The nuclear transcriptional control complex, NFκlB is central in signaling downstream from TNF-α; so we began to study the function of NFκlB in erythroid cells. We made three very interesting initial findings: 1) first we found that NFκlB binding to DNA increased very slowly in HCD57 erythroid cells treated with erythropoietin (EPO, the hormone required for red blood cell development). An inhibitory effect of adding a neutralizing antibody to TNF-α on EPO-stimulated NFκlB DNA suggested this increase in NFκlB was due to TNF-α rather than direct EPO signaling. 2) We also found that NFκlB binding to DNA increased 10-fold or greater during erythroid differentiation. We found greatly increased NFκlB DNA binding in HCD57 cells that differentiated due to over-expression of JunB, F-MEL induced by DMSO, or human UT7-EPO or murine HCD57 cells induced to differentiate with hemin. 3) Surprisingly, we found that the NFκlB DNA binding complex in mouse primary erythroid cells and the erythroid cells lines tested was almost exclusively composed of the atypical p50/p50/Bcl3 NFκlB rather than the canonical p65/p50 or the non-canonical p65/p52 NFκlB. When we begin to study the biological significance of this atypical NFκlB in EPO-mediated erythroid differentiation in vivo using genetic tools, we found marked deficiencies in the development of erythroid cells in either the nfkb1−/− mice (p50−/−) or the bcl3 −/− mice. The nfkb1−/− mice were mildly anemic. The number of red blood cells in the circulation of these mice was statistically lower than in control mice. The number of CFU-e was also reduced in nfkb1−/− mice. Using the Ter-119 and CD71 staining method, we noted that proerythroblasts and immature erythroid cells increased and mature erythroblasts decreased in either non-anemic bone marrow or anemic spleens of nfkb1−/− mice. Forward scatter of Ter-119+ cells also showed an increased size of the average immature erythroid cell in the bone a marrow of nfkb1 −/− mice, suggesting a block in differentiation and continued cell cycling of the immature erythroblasts. Similar erythroid defects were observed in the spleens of anemic bcl-3−/− mice. nfkb1−/− mice and bcl-3−/− mice are also apparently unable to produce new reticulocytes as effectively as wild type mice after induction of anemia. Our working hypothesis is without expression of either p50 or Bcl-3 NFκlB proteins, immature erythroid cells continue to proliferate and ineffectively differentiate. In summary, the atypical p50/p50/Bcl-3 NFκlB complex appears necessary for maximal differentiation of immature erythroid cells.

Integrated Genome-Wide CTCF and CohesinSA1 Occupancy and Expression Analyses in Erythropoiesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1305-1305
Author(s):  
Vincent P Schulz ◽  
Laurie A. Steiner ◽  
Yelena Maksimova ◽  
Patrick G. Gallagher
Keyword(s):  
Cell Types ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Cell Type ◽  
Chromatin Domains ◽  
Cd34 Cells ◽  
Domain Boundaries ◽  
Intergenic Regions ◽  
Cell Type Specific ◽  
In Erythroid Cells

Abstract Abstract 1305 CTCF and cohesion are critical regulators of cellular growth, development and differentiation. CTCF has multiple functions including acting at gene promoters as a transcriptional activator or repressor, mediating long-range chromatin interactions, and acting as a chromatin insulator element. The cohesin complex is also multifunctional, participating in chromosome segregation during cell division, facilitating DNA-promoter interactions through cell-type specific DNA-looping, participating in DNA repair, and participating with CTCF in enhancer blocking. The cohesin complex is composed of 4 proteins Smc1, Smc3, Scc1, and either SA1 or SA2. The presence of SA1 or SA2 is mutually excusive, leading to 2 related, but distinct complexes, cohesinSA1 and cohesin.SA2. The SA1 component of the complex directly interacts with CTCF. To gain insight into how CTCF and cohesin regulate genes in erythroid development, chromatin immunoprecipitation coupled with high throughput sequencing (ChIP-seq) and mRNA transcriptome analyses were performed in human CD34+ hematopoietic stem and progenitor cells and cultured primary human erythroid (R3/R4 stage) cells, the results combined, and the interactomes compared. The MACS program identified 26,330 sites of CTCF and 23,396 sites of cohesinSA1 occupancy in CD34+ and 39,782 sites of CTCF and 33,497 sites of cohesinSA1 occupancy in erythroid cell chromatin (p<10e-5, fold enrichment>5). In CD34+ cells, the majority of CTCF and cohesinSA1 binding sites were located in intergenic regions (56 and 57%,) and introns (33 and 34%). In contrast, in erythroid cells, CTCF and cohesinSA1 binding had migrated to gene promoters (16% vs 2% and 24% vs 2%, respectively) with less binding in intergenic regions and introns. Sites of binding in erythroid cells were similar to that observed in fibroblasts, another differentiated cell-type. CTCF has sites of both cell-type specific and cell-type invariant binding. The Galaxy tool was utilized to compare sites of CTCF occupancy in 7 additional cell types. In CD34+ cells, only 5% sites of CTCF binding were CD34+ cell-type specific. In erythroid cells, 36% of CTCF binding sites were erythroid-specific. These unique sites were located primarily in enhancers and introns and were rarely seen in promoters. Refseq genes within 3kb of erythroid cell-specific CTCF sites were highly significantly enriched for the following GO terms: induction of apoptosis by extracellular signals, cytoskeleton organization, cellular response to stress, and macromolecule catabolic process. In both cell types, RefSeq genes within 3kb of an invariant CTCF site were consistently expressed at lower levels c.f. genes within 3kb of CD34+- or erythroid cell-specific CTCF sites. Analyzing CTCF-cohesinSA1 co-occupancy, there were 17,755 sites of CTCF and cohesinSA1 co-occupancy in CD34+ cells, accounting for 75% of CTCF sites and 67% of cohesinSA1 sites. In erythroid cells, 19,933 sites of occupancy were shared between CTCF and cohesinSA1, representing 50% of CTCF sites and 60% of cohesinSA1 sites. Finally, it has been suggested that CTCF marks chromatin domains in a cell-type specific manner. To determine whether CTCF and cohesinSA1 are present at domain boundaries in erythropoiesis, ChIP-seq for H3K27me3, a repressive chromatin mark, was performed. Chromatin domains were predicted using the Rseg program. 9,480 and 18,511 H3K27me3 chromatin domains were identified in CD34+ and erythroid cells, respectively, with average domain lengths of 31kb in CD34+ and 28kb in erythroid cells. There were 692 and 2,096 CTCF sites that marked domain boundaries in CD34+ and erythroid cells, respectively. These CTCF sites were cell-type specific, as only 75 of these CTCF sites were shared between CD34+ and erythroid cells. In both cell types, the majority of CTCF sites marking domain boundaries were found in distal intergenic regions and introns. CohesinSA1 was also frequently found at domain boundaries, present at 566 and 1830 domain boundaries in CD34+ and erythroid cells, respectively. Co-localization of CTCF with cohesinSA1 at domain boundaries was also common, with 66% of CTCF sites and 58% of CTCF sites binding both CTCF and cohesionSA1 in CD34+ and erythroid cells, respectively. These data indicate that CTCF and cohesin have multiple roles in regulating gene expression in erythropoiesis. Disclosures: No relevant conflicts of interest to declare.

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