Identification of NuRSERY,a new functional HDAC complex composed by HDAC5, GATA1, EKLF and pERK present in human erythroid cells

To clarify the role of HDACs in erythropoiesis, expression, activity and function of class I (HDAC1, HDAC2, HDAC3) and class IIa (HDAC4, HDAC5) HDACs during in vitro maturation of human erythroblasts were compared. During erythroid maturation, expression of HDAC1, HDAC2 and HDAC3 remained constant and activity and GATA1 association (its partner of the NuRD complex), of HDAC1 increased. By contrast, HDAC4 content drastically decreased and HDAC5 remained constant in content but decreased in activity. In erythroid cells, pull down experiments identified the presence of a novel complex formed by HDAC5, GATA1, EKLF and pERK which was instead undetectable in cells of the megakaryocytic lineage. With erythroid maturation, association among HDAC5, GATA1 and EKLF persisted but levels of pERK sharply decreased. Treatment of erythroleukemic cells with inhibitors of ERK phosphorylation reduced by >90% the total and nuclear content of HDAC5, GATA1 and EKLF, suggesting that ERK phosphorylation is required for the formation of this complex. Based on the function of class IIa HDACs as chaperones of other proteins to the nucleus and the erythroid-specificity of HDAC5 localization, this novel HDAC complex was named nuclear remodeling shuttle erythroid (NuRSERY). Exposure of erythroid cells to the class II-selective HDAC inhibitor (HDACi) APHA9 increased γ/(γ+β) globin expression ratios (Mai et al., 2007), suggesting that NuRSERY may regulate globin gene expression. In agreement with this hypothesis, exposure of erythroid cells to APHA9 greatly reduced the association among HDAC5, GATA1 and EKLF. Since exposure to APHA9 did not affect survival rates or p21 activation, NuRSERY may represent a novel, possibly less toxic, target for epigenetic therapies of hemoglobinopaties and other disorders.     

130-kDa smooth muscle myosin light chain kinase is transcribed from a CArG-dependent, internal promoter within the mouse mylk gene

The 130-kDa smooth muscle myosin light chain kinase (smMLCK) is a Ca2+/CaM-regulated enzyme that plays a pivotal role in the initiation of smooth muscle contraction and regulation of cellular migration and division. Despite the critical importance of smMLCK in these processes, little is known about the mechanisms regulating its expression. In this study, we have identified the proximal promoter of smMLCK within an intron of the mouse mylk gene. The mylk gene encodes at least two isoforms of MLCK (130 and 220 kDa) and telokin. Luciferase reporter gene assays demonstrated that a 282-bp fragment (-167 to +115) of the smMLCK promoter was sufficient for maximum activity in A10 smooth muscle cells and 10T1/2 fibroblasts. Deletion of the 16 bp between -167 and -151, which included a CArG box, resulted in a nearly complete loss of promoter activity. Gel mobility shift assays and chromatin immunoprecipitation assays demonstrated that serum response factor (SRF) binds to this CArG box both in vitro and in vivo. SRF knockdown by short hairpin RNA decreased endogenous smMLCK expression in A10 cells. Although the SRF coactivator myocardin induced smMLCK expression in 10T1/2 cells, myocardin activated the promoter only two- to fourfold in reporter gene assays. Addition of either intron 1 or 6 kb of the 5' upstream sequence did not lead to any further activation of the promoter by myocardin. The proximal smMLCK promoter also contains a consensus GATA-binding site that bound GATA-6. GATA-6 binding to this site decreased endogenous smMLCK expression, inhibited promoter activity in smooth muscle cells, and blocked the ability of myocardin to induce smMLCK expression. Altogether, these data suggest that SRF and SRF-associated factors play a key role in regulating the expression of smMLCK.