Epigenetic characterization of hematopoietic stem cell differentiation using miniChIP and bisulfite sequencing analysis

Hematopoietic stem cells (HSC) produce all blood cell lineages by virtue of their capacity to self-renew and differentiate into progenitors with decreasing cellular potential. Recent studies suggest that epigenetic mechanisms play an important role in controlling stem cell potency and cell fate decisions. To investigate this hypothesis in HSC, we have modified the conventional chromatin immunoprecipitation assay allowing for the analysis of 50,000 prospectively purified stem and progenitor cells. Together with bisulfite sequencing analysis, we found that methylated H3K4 and AcH3 and unmethylated CpG dinucleotides colocalize across defined regulatory regions of lineage-affiliated genes in HSC. These active epigenetic histone modifications either accumulated or were replaced by increased DNA methylation and H3K27 trimethylation in committed progenitors consistent with gene expression. We also observed bivalent histone modifications at a lymphoid-affiliated gene in HSC and downstream transit-amplifying progenitors. Together, these data support a model in which epigenetic modifications serve as an important mechanism to control HSC multipotency.     

Sirtuin 1 regulation of developmental genes during differentiation of stem cells

The longevity-promoting NAD⁺–dependent class III histone deacetylase Sirtuin 1 (SIRT1) is involved in stem cell function by controlling cell fate decision and/or by regulating the p53-dependent expression of NANOG. We show that SIRT1 is down-regulated precisely during human embryonic stem cell differentiation at both mRNA and protein levels and that the decrease in Sirt1 mRNA is mediated by a molecular pathway that involves the RNA-binding protein HuR and the arginine methyltransferase coactivator-associated arginine methyltransferase 1 (CARM1). SIRT1 down-regulation leads to reactivation of key developmental genes such as the neuroretinal morphogenesis effectors DLL4, TBX3, and PAX6, which are epigenetically repressed by this histone deacetylase in pluripotent human embryonic stem cells. Our results indicate that SIRT1 is regulated during stem cell differentiation in the context of a yet-unknown epigenetic pathway that controls specific developmental genes in embryonic stem cells.     

Programming smooth muscle plasticity with chromatin dynamics

Smooth muscle cells (SMCs) possess remarkable phenotypic plasticity that allows rapid adaptation to fluctuating environmental cues. For example, vascular SMCs undergo profound changes in their phenotype during neointimal formation in response to vessel injury or within atherosclerotic plaques. Recent studies have shown that interaction of serum response factor (SRF) and its numerous accessory cofactors with CArG box DNA sequences within promoter chromatin of SMC genes is a nexus for integrating signals that influence SMC differentiation in development and disease. During development, SMC-restricted sets of posttranslational histone modifications are acquired within the CArG box chromatin of SMC genes. These modifications in turn control the chromatin-binding properties of SRF. The histone modifications appear to encode a SMC-specific epigenetic program that is used by extracellular cues to influence SMC differentiation, by regulating binding of SRF and its partners to the chromatin template. Thus, SMC differentiation is dynamically regulated by the interplay between SRF accessory cofactors, the SRF-CArG interaction, and the underlying histone modification program. As such, the inherent plasticity of the SMC lineage offers unique glimpses into how cellular differentiation is dynamically controlled at the level of chromatin within the context of changing microenvironments. Further elucidation of how chromatin regulates SMC differentiation will undoubtedly yield valuable insights into both normal developmental processes and the pathogenesis of several vascular diseases that display detrimental SMC phenotypic behavior.     

Epigenetic regulation of key vascular genes and growth factors

The role of small RNAs in epigenetic regulation is an emerging field. This research may also open novel treatment strategies based on manipulation of the epigenetic status of the target tissues. Our objective is to review epigenetic regulation of key vascular genes and growth factors. Vascular endothelial growth factor A (VEGF-A) is one of the key players in regulating and maintaining cardiovascular functions and pathology. Although its epigenetic regulation is still not completely understood, expression of the VEGF gene can be manipulated by epigenetic mechanisms using small RNAs that are targeted to the gene promoter which results in the alteration of histone code. VEGF exerts its effects mostly through two receptors, VEGFR1 and VEGFR2, and their expression is also regulated by promoter DNA methylation in various cancer cells. These findings suggest the importance of epigenetic mechanisms in the regulation of vascular functions.     

Epigenetic histone acetylation modifiers in vascular remodelling: new targets for therapy in cardiovascular disease

Significant progress has been made in the clinical managementof a variety of cardiovascular diseases. Nevertheless, the therapeuticefficacy of the current treatment modalities for atherosclerosisand restenosis is not fully sufficient in a large proportionof patients. One of the major contributing factors is the clinicaland biological heterogeneity of these still life-threateningdiseases, which involve processes that we do not fully understandat the moment. Over the past decades, it has become increasinglyclear that part of the gene–environmental interactionsrelevant for complex diseases is regulated by epigenetic mechanismssuch as histone acetylation and DNA methylation. Epigeneticprocesses modulate gene expression patterns without modifyingthe actual DNA sequence and have profound effects on the cellularrepertoire of expressed genes. They contribute to the expressionof genes that play a key role in extracellular matrix formation,inflammation, and proliferation, processes involved in cardiovascularpathologies such as atherosclerosis and restenosis. Therefore, in this review, we argue that epigenetic regulatorsinvolved in histone acetylating and deacetylating activitiescontribute to the pathogenesis of atherosclerosis and restenosis.Furthermore, as alterations in chromatin structure are reversible,these epigenetic modifications are amendable to pharmacologicalintervention, which may prove to be an effective treatment modalityfor the management of cardiovascular diseases.     

Global modulation of chromatin dynamics mediated by dephosphorylation of linker histone H1 is necessary for erythroid differentiation

Differentiation of metazoan cells involves dramatic changes in gene expression patterns and proliferative capacity driven primarily by epigenetic mechanisms. Here we used in vivo photobleaching techniques and biochemical assays to investigate the contribution of alterations in chromatin dynamics to the differentiation of murine erythroleukemia (MEL) cells, a model system for erythroid development. As MEL cells differentiate the binding affinity of all linker histone variants increases, indicative of an overall decrease in chromatin flexibility. Changes in H1(0) binding properties depend on phosphorylation at one or more of three cyclin-dependent kinase sites. The presence of constructs mimicking constitutively phosphorylated H1 results in a significant inhibition in the acquisition of commitment to terminal cell division and the expression of erythroid-specific properties. These data indicate that the progressive loss of cdk activity associated with MEL cell differentiation leads to the accumulation of dephosphorylated linker histones and restricted chromatin flexibility and that these are necessary events in the progression of erythroid differentiation. We present additional data indicating that the presence of phosphorylated H1 has a dominant effect on the binding behavior of other linker histones and propose a model for the role of linker histone phosphorylation in which these modifications act within the context of assembled chromatin.