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.

Ibrutinib induces chromatin reorganisation of chronic lymphocytic leukaemia cells.

Chronic lymphocytic leukaemia (CLL) is the most common leukaemia in Western countries. It has recently been shown that the homogeneity of the chromatin landscape between CLL cells contrasts with the important observed genetic heterogeneity of the disease. To gain further insight into the consequences of disease evolution on the epigenome's plasticity, we monitored changes in chromatin structure occurring in vivo in CLL cells from patients receiving continuous Ibrutinib treatment. Ibrutinib, an oral inhibitor of the Bruton's tyrosine kinase (BTK) has proved to be remarkably efficient against treatment naïve (TN), heavily pre-treated and high-risk chronic lymphocytic leukaemia (CLL), with limited adverse events. We established that the chromatin landscape is significantly and globally affected in response to Ibrutinib. However, we observed that prior to treatment, CLL cells show qualitative and quantitative variations in chromatin structure correlated with both EZH2 protein level and cellular response to external stimuli. Then, under prolonged exposure to Ibrutinib, a loss of the two marks associated with lysine 27 (acetylation and trimethylation) was observed. Altogether, these data indicate that the epigenome of CLL cells from the peripheral blood change dynamically in response to stimuli and suggest that these cells might adapt to the Ibrutinib "hit" in a process leading toward a possible reduced sensitivity to treatment.

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.

A NF-κB-dependent dual promoter-enhancer initiates the lipopolysaccharide-mediated transcriptional activation of the chicken lysozyme in macrophages.

The transcriptional activation of the chicken lysozyme gene (cLys) by lipopolysaccharide (LPS) in macrophages is dependent on transcription of a LPS-Inducible Non-Coding RNA (LINoCR) triggering eviction of the CCCTC-binding factor (CTCF) from a negative regulatory element upstream of the lysozyme transcription start site. LINoCR is transcribed from a promoter originally characterized as a hormone response enhancer in the oviduct. Herein, we report the characterization of this cis-regulatory element (CRE). In activated macrophages, a 60 bp region bound by NF-κB, AP1 and C/EBPß controls this CRE, which is strictly dependent on NF-κB binding for its activity in luciferase assays. Moreover, the serine/threonine kinase IKKα, known to be recruited by NF-κB to NF-κB-dependent genes is found at the CRE and within the transcribing regions of both cLys and LINoCR. Such repartition suggests a simultaneous promoter and enhancer activity of this CRE, initiating cLys transcriptional activation and driving CTCF eviction. This recruitment was transient despite persistence of both cLys transcription and NF-κB binding to the CRE. Finally, comparing cLys with other LPS-inducible genes indicates that IKKα detection within transcribing regions can be correlated with the presence of the elongating form of RNA polymerase II or concentrated in the 3' end of the gene.

Heterochromatin protein 1 gamma and IκB kinase alpha interdependence during tumour necrosis factor gene transcription elongation in activated macrophages.

IκB kinase α (IKKα) is part of the cytoplasmic IKK complex regulating nuclear factor-κB (NF-κB) release and translocation into the nucleus in response to pro-inflammatory signals. IKKα can also be recruited directly to the promoter of NF-κB-dependent genes by NF-κB where it phosphorylates histone H3 at serine 10, triggering recruitment of the bromodomain-containing protein 4 and the positive transcription elongation factor b. Herein, we report that IKKα travels with the elongating form of ribonucleic acid polymerase II together with heterochromatin protein 1 gamma (HP1γ) at NF-κB-dependent genes in activated macrophages. IKKα binds to and phosphorylates HP1γ, which in turn controls IKKα binding to chromatin and phosphorylation of the histone variant H3.3 at serine 31 within transcribing regions. Downstream of transcription end sites, IKKα accumulates with its inhibitor the CUE-domain containing protein 2, suggesting a link between IKKα inactivation and transcription termination.