Alternative splicing and allosteric regulation modulate the chromatin binding of UHRF1.

UHRF1 is an important epigenetic regulator associated with apoptosis and tumour development. It is a multidomain protein that integrates readout of different histone modification states and DNA methylation with enzymatic histone ubiquitylation activity. Emerging evidence indicates that the chromatin-binding and enzymatic modules of UHRF1 do not act in isolation but interplay in a coordinated and regulated manner. Here, we compared two splicing variants (V1, V2) of murine UHRF1 (mUHRF1) with human UHRF1 (hUHRF1). We show that insertion of nine amino acids in a linker region connecting the different TTD and PHD histone modification-binding domains causes distinct H3K9me3-binding behaviour of mUHRF1 V1. Structural analysis suggests that in mUHRF1 V1, in contrast to V2 and hUHRF1, the linker is anchored in a surface groove of the TTD domain, resulting in creation of a coupled TTD-PHD module. This establishes multivalent, synergistic H3-tail binding causing distinct cellular localization and enhanced H3K9me3-nucleosome ubiquitylation activity. In contrast to hUHRF1, H3K9me3-binding of the murine proteins is not allosterically regulated by phosphatidylinositol 5-phosphate that interacts with a separate less-conserved polybasic linker region of the protein. Our results highlight the importance of flexible linkers in regulating multidomain chromatin binding proteins and point to divergent evolution of their regulation.

DAPK3 participates in the mRNA processing of immediate early genes in chronic lymphocytic leukaemia.

Cross-linking of the B-cell receptor (BCR) induces transcriptional activation of immediate early genes (IEGs) including EGR1 and DUSP2 in chronic lymphocytic leukaemia (CLL). Here, we have shown that this transcriptional activation correlated with histone H3 threonine 6 and 11 phosphorylation. Both transcription and histone post-translational modifications are repressed by ibrutinib, a small molecule inhibitor used in CLL treatment. Moreover, we have identified the death-associated protein kinase 3 (DAPK3), as the kinase mediating these histone phosphorylation marks in response to activation of the BCR signalling pathway with this kinase being recruited to RNA polymerase II in an anti-IgM-dependent manner. DAPK inhibition mimics ibrutinib-induced repression of both IEG mRNA and histone H3 phosphorylation and has anti-proliferative effect comparable to ibrutinib in CLL in vitro. DAPK inhibitor does not repress transcription itself but impacts on mRNA processing and has a broader anti-tumour effect than ibrutinib, by repressing both anti-IgM- and CD40L-dependent activation.

Oxidative stress signaling to chromatin in health and disease.

Oxidative stress has a significant impact on the development and progression of common human pathologies, including cancer, diabetes, hypertension and neurodegenerative diseases. Increasing evidence suggests that oxidative stress globally influences chromatin structure, DNA methylation, enzymatic and non-enzymatic post-translational modifications of histones and DNA-binding proteins. The effects of oxidative stress on these chromatin alterations mediate a number of cellular changes, including modulation of gene expression, cell death, cell survival and mutagenesis, which are disease-driving mechanisms in human pathologies. Targeting oxidative stress-dependent pathways is thus a promising strategy for the prevention and treatment of these diseases. We summarize recent research developments connecting oxidative stress and chromatin regulation.

Dynamic and flexible H3K9me3 bridging via HP1ß dimerization establishes a plastic state of condensed chromatin.

Histone H3 trimethylation of lysine 9 (H3K9me3) and proteins of the heterochromatin protein 1 (HP1) family are hallmarks of heterochromatin, a state of compacted DNA essential for genome stability and long-term transcriptional silencing. The mechanisms by which H3K9me3 and HP1 contribute to chromatin condensation have been speculative and controversial. Here we demonstrate that human HP1ß is a prototypic HP1 protein exemplifying most basal chromatin binding and effects. These are caused by dimeric and dynamic interaction with highly enriched H3K9me3 and are modulated by various electrostatic interfaces. HP1ß bridges condensed chromatin, which we postulate stabilizes the compacted state. In agreement, HP1ß genome-wide localization follows H3K9me3-enrichment and artificial bridging of chromatin fibres is sufficient for maintaining cellular heterochromatic conformation. Overall, our findings define a fundamental mechanism for chromatin higher order structural changes caused by HP1 proteins, which might contribute to the plastic nature of condensed chromatin.

Isoform 1 of TPD52 (PC-1) promotes neuroendocrine transdifferentiation in prostate cancer cells.

The tumour protein D52 isoform 1 (PC-1), a member of the tumour protein D52 (TPD52) protein family, is androgen-regulated and prostate-specific expressed. Previous studies confirmed that PC-1 contributes to malignant progression in prostate cancer with an important role in castration-resistant stage. In the present work, we identified its impact in mechanisms leading to neuroendocrine (NE) transdifferentiation. We established for long-term PC-1 overexpression an inducible expression system derived from the prostate carcinoma cell line LNCaP. We observed that PC-1 overexpression itself initiates characteristics of neuroendocrine cells, but the effect was much more pronounced in the presence of the cytokine interleukin-6 (IL-6). Moreover, to our knowledge, this is the first report that treatment with IL-6 leads to a significant upregulation of PC-1 in LNCaP cells. Other TPD52 isoforms were not affected. Proceeding from this result, we conclude that PC-1 overexpression enhances the IL-6-mediated differentiation of LNCaP cells into a NE-like phenotype, noticeable by morphological changes and increased expression of typical NE markers, like chromogranin A, synaptophysin or beta-3 tubulin. Immunofluorescent staining of IL-6-treated PC-1-overexpressing LNCaP cells indicates a considerable PC-1 accumulation at the end of the long-branched neuron-like cell processes, which are typically formed by NE cells. Additionally, the experimentally initiated NE transdifferentiation correlates with the androgen receptor status, which was upregulated additively. In summary, our data provide evidence for an involvement of PC-1 in NE transdifferentiation, frequently associated with castration resistance, which is a major therapeutic challenge in the treatment of advanced prostate cancer.

CTCF depletion alters chromatin structure and transcription of myeloid-specific factors.

Differentiation is a multistep process tightly regulated and controlled by complex transcription factor networks. Here, we show that the rate of differentiation of common myeloid precursor cells increases after depletion of CTCF, a protein emerging as a potential key factor regulating higher-order chromatin structure. We identified CTCF binding in the vicinity of important transcription factors regulating myeloid differentiation and showed that CTCF depletion impacts on the expression of these genes in concordance with the observed acceleration of the myeloid commitment. Furthermore, we observed a loss of the histone variant H2A.Z within the selected promoter regions and an increase in non-coding RNA transcription upstream of these genes. Both abnormalities suggest a global chromatin structure destabilization and an associated increase of non-productive transcription in response to CTCF depletion but do not drive the CTCF-mediated transcription alterations of the neighbouring genes. Finally, we detected a transient eviction of CTCF at the Egr1 locus in correlation with Egr1 peak of expression in response to lipopolysaccharide (LPS) treatment in macrophages. This eviction is also correlated with the expression of an antisense non-coding RNA transcribing through the CTCF-binding region indicating that non-coding RNA transcription could be the cause and the consequence of CTCF eviction.