ALBERT

Description: Anemia is the predominant clinical manifestation of myelodysplastic syndromes (MDS). Loss or deletion of chromosome 7 is commonly seen in MDS and leads to a poor prognosis. However, the identity of functionally relevant, dysplasia-causing, genes on 7q remains unclear. Dedicator of cytokinesis 4 (DOCK4) is a GTPase exchange factor, and its gene maps to the commonly deleted 7q region. We demonstrate that DOCK4 is underexpressed in MDS bone marrow samples and that the reduced expression is associated with decreased overall survival in patients. We show that depletion of DOCK4 levels leads to erythroid cells with dysplastic morphology both in vivo and in vitro. We established a novel single-cell assay to quantify disrupted F-actin filament network in erythroblasts and demonstrate that reduced expression of DOCK4 leads to disruption of the actin filaments, resulting in erythroid dysplasia that phenocopies the red blood cell (RBC) defects seen in samples from MDS patients. Reexpression of DOCK4 in −7q MDS patient erythroblasts resulted in significant erythropoietic improvements. Mechanisms underlying F-actin disruption revealed that DOCK4 knockdown reduces ras-related C3 botulinum toxin substrate 1 (RAC1) GTPase activation, leading to increased phosphorylation of the actin-stabilizing protein ADDUCIN in MDS samples. These data identify DOCK4 as a putative 7q gene whose reduced expression can lead to erythroid dysplasia.

Description: Key Points CUX1 is a transcription factor encoded on a region of chromosome 7 that is frequently deleted in high-risk acute myeloid leukemia. Haploinsufficiency of CUX1/cut promotes hematopoietic overgrowth in both Drosophila melanogaster and human xenograft mouse models in vivo.

Description: The human alpha globin genes are controlled by DNase hypersensitive sites (HS) HS-4, HS-8, HS-10, HS-33 and HS-40 upstream of the ζ gene. Among these, HS40 functions as a strong enhancer of the alpha like genes. The alpha globin genes are situated amidst actively transcribing genes, but are transcriptionally silent in non-erythroid cells including hematopoietic progenitor cells We have undertaken an analysis of the chromatin structure of the alpha globin locus, recruitment of transcription factors, and the transcriptional activity of the locus in CD34+ hematopoietic progenitor cells and upon their differentiation into erythroid cells. Chromatin immunoprecipitation (ChIP) followed by PCR analysis of all the regulatory and structural segments of the α-globin locus were performed using antibodies against chemically modified tails of histone H3, the insulator binding factor CTCF, transcription factors such as GATA-1 and NF-E2, and Pol II. Both H3Me2K4 and H3AcK9 modifications were present at HS48 and HS33 in CD34+ cells and substantially increase when these cells are differentiated into erythroid lineage. At the HS40 region, these modifications were present at a low level in CD34+ cells and did not change during erythroid differentiation. Among the α-like gene promoters, we find these modifications at the Mu and theta gene promoters in CD34+ cells and they increase during erythropoiesis. These modifications were absent at the zeta gene promoter consistent with the inactivity of this gene during definitive erythropoiesis. Overall the dominant HS40 enhancer possesses moderate levels of H3Me2K4 and H3AcK9 modifications, and its cognate major a-globin promoter is devoid of these modifications in CD34+ cells even when these cells are differentiated into erythroid lineage. The entire α-globin locus including the HS enhancer regions and a-like gene promoters did not contain the unphosphorylated (initiation) form of Pol II recruitment in CD34+ cells. When these cells differentiated into the erythroid lineage, Pol II was recruited at the HS40 and HS48 regions and at the Mu and theta promoters. Rearrangement of the CTCF binding sites at the α-globin locus occurs during differentiation of CD34+ cells into the erythroid lineage. In CD34+ cells, as in HeLa cells, the α-globin genes are flanked by multiple CTCF binding events at the 5′ and 3′ ends of the locus. At the 5′ end of the locus, the HS40 and HS48 sequences were surrounded by four CTCF binding sites at HS33, HS46, HS55 and HS90. At the 3′ end of the locus CTCF was observed at the theta globin promoter and at the 3′ end of the theta globin gene. Upon differentiation of the CD34+ cells into the erythroid pathway, CTCF recruitment is significantly reduced at HS90 and HS46 sequences, while the sites at HS55 and HS33 show increased CTCF binding. Thus, in contrast to the CD34+ cells, the HS40 and HS48 sequences are y flanked by two CTCF recruitment sites in erythroid cells. Such a differential placement of CTCF binding sites suggests that differential interaction among CTCF sites may regulate the effects of the HS-40 enhancer. In erythroid cells, a strong HS40 enhancer formed by virtue of the recruitment of the enhancer factors can overcome blocking by the downstream flanking CTCF site and this might be mediated by specific interactions between the two flanking insulators. The CTCF binding at the 3′ end of the theta globin gene is abolished during erythropoiesis of CD34+ cells. However, the recruitment of CTCF at the theta globin promoter is unchanged suggesting that the theta globin may be insulated by the influence of the α-globin enhancer sequences. We have detected transcripts from parts of the theta and zeta genes and intergenic regions in HeLa, NB4 and 06990 lymphoblastoid cells and primary erythroid cells in culture. The transcription of the locus was localized to certain regions, suggesting that there may be unappreciated transcriptional regulatory elements within the locus.

Description: The human beta globin locus control region (LCR) aids in the establishment of an open chromatin configuration thereby facilitating the expression of linked downstream globin genes in a temporal and spatial manner along the developmental ontogeny. The LCR also functions as a classical enhancer of the beta like globin genes. The four DNAase-I hypersensitive regions (HS 1–4) on the LCR are believed to act as nucleation centers for the assembly of multi-protein transcriptional complexes that interact with the distant gene promoters. Present work is focused on characterizing the protein complex assemblies on the HS4 region and delineating their function in the context of goblin gene regulation. Our finding shows an assembly of a 2–5 MDa complex on the HS4 locus that is both erythroid restricted and sequence specific. Electrophoretic mobility shift assays (EMSA) were performed using K562 and HeLa nuclear extracts and ∼35mer double stranded oligonucleotides with 7 bp overlaps tiling the entire core HS4 sequence. We detected prominent K562 specific bands formed with an AT rich 39 bp HS4 sequence. Sequence specificity of DNA-protein complex formation on this AT rich sequence was determined by competitive EMSA assays. Gel supershift assays with a wide range of antibodies and subsequent partial purification of this multiprotein complex followed by the LC-MS/MS analysis reveal the presence of a mini chromosome maintenance (MCM) complex that is associated with the DNA replication, DNA repair associated Rad50-Mre-11-NBS-1 complex, histone acetyl transferase p300 and DNA binding transcription factors ILF2 (NF45) and ILF3 (NF90). The association of these proteins was determined by co-immunoprecipitation experiments. Chromatin immunoprecipitation (ChIP) experiments demonstrate the in vivo recruitment of these proteins on the HS4 region of the LCR. We are currently working on further characterization and functional studies of this complex and its significance on the role of the LCR in DNA replication and repair.

Description: Abstract 1012 The clinical symptoms of sickle cell disease can be ameliorated by increased fetal hemoglobin (HbF) levels. Previous work from our laboratory demonstrated that Trichostatin A (TSA) and sodium butyrate (NaB) activate γ-globin expression via p38 MAPK signaling. In addition, cAMP response element binding protein 1 (CREB1) was shown to trans-activate the -1222 Gγ-globin cAMP response element (G-CRE) in a transient assay system. To study the role of p38 MAPK signaling in γ-globin regulation, loss of function siRNA studies were performed in K562 cells. siRNA-mediated knockdown of p38 MAPK resulted in 72% loss of γ-globin transcription. Furthermore, enforced stable expression of MKK3/6 increased the phosphorylated form of p38 MAPK by 70%, which in turn produced a 2- to 3-fold increase in γ-globin mRNA and HbF levels. Likewise, siCREB1 treatment reduced CREB1 levels by 62% and down regulated γ-globin expression 59%. In the same vein, stable expression of recombinant CREB1 activated HbF by 2-fold. These findings were subsequently confirmed in human primary erythroid cells grown in a two-phase liquid culture system. On day 11, we observed 50–70% γ-globin silencing after CREB1 and p38 MAPK siRNA knockdown with 60% target gene silencing. CREB1 enforced expression trans-activated γ-globin 4.5-fold which was accompanied by a 90% increase in HbF-FITC positive cells and HbF levels. Collectively, these data demonstrate that p38 MAPK and CREB1 are required for steady-state γ-globin gene transcription. To determine the role of the G-CRE in γ-globin regulation, the Gγ-globin promoter (-1500 to +36) was cloned into pGL4.17 Luc2/neo to produce pGγLuc2 (wild type) and mutant plasmids: -1225 G/A (m1), -1227 AC/TG (m2) and a scrambled G-CRE (m3s). Five K562 stable lines including KLuc2 (promoterless), KGγLuc2, KGγLuc2(m1), KGγLuc2(m2) and KGγLuc2(m3s) were established. Luciferase activity in KGγLuc2 was 1000-fold higher than in the control Kluc2 line; all mutations produced 〉90% loss of luciferase activity and a loss of γ-globin trans-activation by TSA and NaB. Next, siRNA studies were completed to determine if the G-CRE is required for γ-globin activation. A dose-dependent loss of promoter activity was observed after p38 MAPK and CREB1 siRNA knockdown of the KGγLuc2 cell line; however, promoter silencing was not observed in the mutant lines supporting a role for the G-CRE in p38 MAPK/CREB1 mediated γ-globin regulation. To study in vivo binding, chromatin immunoprecipitation (ChIP) assays were performed with CREB1 antibody in the KGγLuc2 stable line. We observed comparable 2- to 3-fold chromatin enrichment with CREB1 compared to the control IgG in the G-CRE regions of the pGγLuc2 plasmid and endogenous Gγ-globin promoter. To determine if an enhanceosome complex is bound to the G-CRE, we performed affinity column pull-down/mass spectrometry analysis. K562 nuclear extract was purified on a Heparin Sepharose column, following which fractions eluting at 0.6M NaCl showing peak gel shift binding activities with the G-CRE oligo were loaded into a size selecting Suprose 6 gel exclusion column. G-CRE eluting fractions were then identified by protein microsequencing (MS/MS). We identified CREB1, ATF2, c-Jun, BRG-1, hnRNPC1/C2, and the TCP-1 complex as major components. To determine protein co-localization, promoter pull-down assays were performed using biotinylated wild type and mutant (AC/TG) G-CRE probes and K562 nuclear extracts. We observed simultaneous CREB1, ATF-2 and cJun binding to the G-CRE which was abolished in the mutant probe. However, Brg1 was bound after NaB (2mM) induction. Subsequent co-IP studies showed interactions between ATF-2 and Brg1, CREB1, cJun, and hnRNPC1C2, which was further confirmed by co-elution profile of these molecules observed by sucrose gradient centrifugation, thus implying association as one complex. These data support complex protein-protein interactions in the G-CRE, which modulate γ-globin gene expression. Additional studies will be performed in primary erythroid cells using siRNA-based gene silencing and ChIP assays to determine novel mechanisms of γ-globin regulation and to define in vivo binding of proteins identified in the G-CRE enhanceosome complex. Disclosures: No relevant conflicts of interest to declare.

Description: Pregnancy in humans is a multi-step complex physiological process comprising three discrete events, decidualization, implantation and placentation. Its overall success depends on the incremental advantage that each of the preceding stages passes on to the next. The success of these synchronized sequels of events is an outcome of timely coordination between them. The pregnancy events are coordinated and governed primarily by the ovarian steroid hormones, estrogen and progesterone, which are essentially ligand-activated transcription factors. It’s well known that intercellular signaling of steroid hormones engages a plethora of adapter proteins that participate in executing the biological functions. This involves binding of the hormone receptor complex to the DNA response elements in a sequence specific manner. Working with Drosophila melanogaster, the heat shock proteins (HSPs) were originally described by Ferruccio Ritossa back in the early 1960s. Over the years, there has been considerable advancement of our understanding of these conserved families of proteins, particularly in pregnancy. Accumulating evidence suggests that endometrial and uterine cells have an abundance of HSP27, HSP60, HSP70 and HSP90, implying their possible involvement during the pregnancy process. HSPs have been found to be associated with decidualization, implantation and placentation, with their dysregulation associated with implantation failure, pregnancy loss and other feto-maternal complications. Furthermore, HSP is also associated with stress response, specifically in modulating the ER stress, a critical determinant for reproductive success. Recent advances suggest a therapeutic role of HSPs proteins in improving the pregnancy outcome. In this review, we summarized our latest understanding of the role of different members of the HSP families during pregnancy and associated complications based on experimental and clinical evidences, thereby redefining and exploring their novel function with new perspective, beyond their prototype role as molecular chaperones.