We have previously described a multiprotein complex termed the BHC or BRAF-HDAC complex, which is required for the repression of neuronal-specific genes. We have shown that the BHC complex is recruited by a neuronal silencer, REST (RE1-silencing transcription factor), and mediates the repression of REST-responsive genes. BHC is a multiprotein complex consisting of two enzymatic activities: a histone deacetylase (HDAC1 or 2) and a recently described histone demethylase (BHC110, also known as LSD1 or AOF2). Here we show that BHC110-containing complexes show a nearly fivefold increase in demethylation of histone H3 lysine 4 (H3K4) compared to recombinant BHC110. Furthermore, recombinant BHC110 is unable to demethylate H3K4 on nucleosomes, but BHC110-containing complexes readily demethylate nucleosomes. In vitro reconstitution of the BHC complex using recombinant subunits reveals an essential role for the REST corepressor CoREST, not only in stimulating demethylation on core histones but also promoting demethylation of nucleosomal substrates. We find that nucleosomal demethylation is the result of CoREST enhancing the association between BHC110 and nucleosomes. Depletion of CoREST in in vivo cell culture results in de-repression of REST-responsive gene expression and increased methylation of H3K4. Together, these results highlight an essential role for CoREST in demethylation of H3K4 both in vitro and in vivo.

Demethylation of histone H3 lysine 4 is carried out by BHC110/LSD1, an enzyme with close homology to monoamine oxidases (MAO). Monoamine oxidase A or B are frequent targets of selective and nonselective small molecular inhibitors used for treatment of depression. Here we show that in contrast to selective monoamine oxidase inhibitors such as pargyline, nonselective monoamine oxidase inhibitors potently inhibit nucleosomal demethylation of histone H3 lysine 4. Tranylcypromine (brand name Parnate) displayed the best inhibitory activity with an IC50 of less than 2 microM. Treatment of P19 embryonal carcinoma cells with tranylcypromine resulted in global increase in H3K4 methylation as well as transcriptional derepression of two BHC110 target genes, Egr1 and the pluripotent stem cell marker Oct4. These results attest to the effectiveness of tranylcypromine as a small molecular inhibitor of histone demethylation.

Cultured human embryonic stem (hES) cells can acquire genetic and epigenetic changes that make them vulnerable to transformation. As hES cells with cancer-cell characteristics share properties with normal hES cells, such as self-renewal, teratoma formation and the expression of pluripotency markers, they may be misconstrued as superior hES cells with enhanced 'stemness'. We characterize two variant hES cell lines (v-hESC-1 and v-hESC-2) that express pluripotency markers at high levels and do not harbor chromosomal abnormalities by standard cytogenetic measures. We show that the two lines possess some features of neoplastic progression, including a high proliferative capacity, growth-factor independence, a 9- to 20-fold increase in frequency of tumor-initiating cells, niche independence and aberrant lineage specification, although they are not malignant. Array comparative genomic hybridization reveals an amplification at 20q11.1-11.2 in v-hESC-1 and a deletion at 5q34a-5q34b;5q3 and a mosaic gain of chromosome 12 in v-hESC-2. These results emphasize the need for functional characterization to distinguish partially transformed and normal hES cells.

Histone deacetylase (HDAC) inhibitors are a promising class of anticancer agents for the treatment of solid and hematological malignancies. The precise mechanism by which HDAC inhibitors mediate their effects on tumor cell growth, differentiation, and/or apoptosis is the subject of intense research. Previously we described a family of multiprotein complexes that contain histone deacetylase 1/2 (HDAC1/2) and the histone demethylase BHC110 (LSD1). Here we show that HDAC inhibitors diminish histone H3 lysine 4 (H3K4) demethylation by BHC110 in vitro. In vivo analysis revealed an increased H3K4 methylation concomitant with inhibition of nucleosomal deacetylation by HDAC inhibitors. Reconstitution of recombinant complexes revealed a functional connection between HDAC1 and BHC110 only when nucleosomal substrates were used. Importantly, while the enzymatic activity of BHC110 is required to achieve optimal deacetylation in vitro, in vivo analysis following ectopic expression of an enzymatically dead mutant of BHC110 (K661A) confirmed the functional cross talk between the demethylase and deacetylase enzymes. Our studies not only reveal an intimate link between the histone demethylase and deacetylase enzymes but also identify histone demethylation as a secondary target of HDAC inhibitors.

The histone demethylase lysine demethylase 5b (KDM5b) specifically demethylates lysine 4 of histone H3 (meH3K4), thereby repressing gene transcription. KDM5b regulates cell cycle control genes in cancer and is expressed in the early epiblast. This suggests that KDM5b plays a developmental role by maintaining uncommitted progenitors. Here we show that transient overexpression of KDM5b in embryonic stem cells decreases the expression of at least three different modulators of cell fate decisions, Egr1, p27 KIP1 , and BMI1, by demethylation of their promoters. Constitutively increased KDM5b expression results in an increased mitotic rate and a decreased global 3meH3K4 but no change in cell identity. Results of two separate differentiation assays, neural differentiation and embryoid body EB (EB) formation, showed that KDM5b reduced the terminally differentiated cells and increased proliferating progenitors. These were achieved by two mechanisms, blocking of the upregulation of cell lineage markers and maintenance of cyclins, that allowed cells to escape differentiation and remain uncommitted. Additionally, EBs maintain high levels of Oct4 and Nanog and can be dissociated to reestablish highly proliferative cultures. The persistence of uncommitted progenitors may be due to the direct regulation of the Tcf/Lef family member mTcf3/hTcf7L1, an upstream regulator of Nanog expression. These findings demonstrate a role for KDM5b in the choice between proliferation and differentiation during development.Transcriptional control is a dynamic process, during which several different histone residues are modified to change RNA polymerase's ability to access the transcriptional start site (19,42). A key component in this process is the methylation of histone H3 lysine 4 (H3K4). Methylation of H3K4 is a key regulator of RNA polymerase binding to active genes (41) and of transcription factor binding within promoter elements (43). The ability of this epigenetic mark to control multiple points in transcription suggests that modulation of H3K4 methylation plays a role in both the activation and the repression of genes.A key aspect of H3K4 methylation is how this epigenetic mark is removed, thereby reducing RNA polymerase's localization to the specific genes. This loss of methyl H3K4

Differentiation of progenitor cells into post-mitotic neurons requires the engagement of mechanisms by which the repressive effects of the neuronal silencer, RE-1 silencing transcription factor (REST), can be overcome. Previously, we described a high-mobility group (HMG)-containing protein, BRAF35, which is a component of a co-repressor complex that is required for the repression of REST-responsive genes. Here, we show that the BRAF35 family member inhibitor of BRAF35 (iBRAF) activates REST-responsive genes through the modulation of histone methylation. In contrast to BRAF35, iBRAFexpression leads to the abrogation of REST-mediated transcriptional repression and the resultant activation of neuronal-specific genes. Analysis of P19 cells during neuronal differentiation revealed an increased concentration of iBRAF at the promoter of neuronal-specific genes coincident with augmented expression of synapsin, recruitment of the methyltransferase MLL and enhanced trimethylation of histone H3 lysine 4 (H3K4). Importantly, ectopic expression of iBRAF is sufficient to induce neuronal differentiation through recruitment of MLL, resulting in increased histone H3K4 trimethylation and activation of neuronal-specific genes. Moreover, depletion of iBRAF abrogates recruitment of MLL and enhancement of histone H3K4 trimethylation. Together, these results indicate that the HMG-domain protein iBRAF has a key role in the initiation of neuronal differentiation.

Genetic analysis in mice has most commonly employed two general strategies: phenotypic screens for spontaneous or induced mutations and genotypic analysis using homologous recombination or gene trapping to produce deletion or insertion mutants. Here we use bacterial artificial chromosome (BAC)-mediated gene-dosage analysis in transgenic mice to reveal novel genetic functions that are not evident from conventional loss-of-function mutations. We demonstrate a role for the zinc-finger transcription factor Zipro1 (formerly Ru49 and Zfp38) in the proliferation of granule cell precursors in the developing cerebellum, and document the contribution of this process to the final stages of cerebellar morphogenesis. We also show that Zipro1 is expressed in skin, and increased Zipro1 dosage results in a hair-loss phenotype associated with increased epithelial cell proliferation and abnormal hair follicle development.

BackgroundHistone deacetylases (HDACs) are enzymes that modulate gene expression and cellular processes by deacetylating histones and non-histone proteins. While small molecule inhibitors of HDAC activity (HDACi) are used clinically in the treatment of cancer, pre-clinical treatment models suggest they also exert neuroprotective effects and stimulate neurogenesis in neuropathological conditions. However, the direct effects of HDACi on cell cycle progression and proliferation, two properties required for continued neurogenesis, have not been fully characterized in adult neural stem cells (NSCs). In this study, we examined the effects of two broad class I and class II HDACi on adult mouse NSCs, the hydroxamate-based HDACi suberoylanilide hydroxamic acid (vorinostat, SAHA) and the short chain fatty acid HDACi sodium butyrate.ResultsWe show that both HDACi suppress the formation of neurospheres by adult mouse NSCs grown in proliferation culture conditions in vitro. DNA synthesis is significantly inhibited in adult mouse NSCs exposed to either SAHA or sodium butyrate and inhibition is associated with an arrest in the G1 phase of the cell cycle. HDACi exposure also resulted in transcriptional changes in adult mouse NSCs. Cdk inhibitor genes p21 and p27 transcript levels are increased and associated with elevated H3K9 acetylation levels at proximal promoter regions of p21 and p27. mRNA levels for notch effector Hes genes and Spry-box stem cell transcription factors are downregulated, whereas pro-neural transcription factors Neurog1 and Neurod1 are upregulated. Lastly, we show HDAC inhibition under proliferation culture conditions leads to long-term changes in cell fate in adult mouse NSCs induced to differentiate in vitro.ConclusionSAHA and sodium butyrate directly regulate cdk inhibitor transcription to control cell cycle progression in adult mouse NSCs. HDAC inhibition results in G1 arrest in adult mouse NSCs and transcriptional changes associated with activation of neuronal lineage commitment programs and a reduction of stem/progenitor state. Changes in differentiated cell state in adult mouse NSCs treated with HDACi under proliferation culture conditions suggests an intrinsic relationship between multipotency, cell cycle progression and HDAC activity in these cells.