TACC3 mediates the association of MBD2 with histone acetyltransferases and relieves transcriptional repression of methylated promoters

We have recently reported that a novel MBD2 interactor (MBDin) has the capacity to reactivate transcription from MBD2-repressed methylated promoters even in the absence of demethylation events. Here we show that another unrelated protein, TACC3, displays a similar activity on methylated genes. In addition the data reported here provide possible molecular mechanisms for the observed phenomenon. Immunoprecipitation experiments showed that MBD2/TACC3 form a complex in vivo with the histone acetyltransferase pCAF. MBD2 could also associate with HDAC2, a component of MeCP1 repression complex. However, we found that the complexes formed by MBD2 with TACC3/pCAF and with HDAC2 were mutually exclusive. Moreover, HAT enzymatic assays demonstrated that HAT activity associates with MBD2 in vivo and that such association significantly increased when TACC3 was over-expressed. Overall our findings suggest that TACC3 can be recruited by MBD2 on methylated promoters and is able to reactivate transcription possibly by favoring the formation of an HAT-containing MBD2 complex and, thus, switching the repression potential of MBD2 in activation even prior to eventual demethylation

Mechanism of retinoic acid-induced transcription: histone code, DNA oxidation and formation of chromatin loops

Histone methylation changes and formation of chro- matin loops involving enhancers, promoters and 3′ end regions of genes have been variously associ- ated with active transcription in eukaryotes. We have studied the effect of activation of the retinoic A re- ceptor, at the RARE–promoter chromatin of CASP9 and CYP26A1 genes, 15 and 45 min following RA ex- posure, and we found that histone H3 lysines 4 and 9 are demethylated by the lysine-specific demethylase, LSD1 and by the JMJ-domain containing demethy- lase, D2A. The action of the oxidase (LSD1) and a dioxygenase (JMJD2A) in the presence of Fe++ elic- its an oxidation wave that locally modifies the DNA and recruits the enzymes involved in base and nu- cleotide excision repair (BER and NER). These events are essential for the formation of chromatin loop(s) that juxtapose the RARE element with the 5′ tran- scription start site and the 3′ end of the genes. The RARE bound-receptor governs the 5′ and 3′ end se- lection and directs the productive transcription cycle of RNA polymerase. These data mechanistically link chromatin loops, histone methylation changes and localized DNA repair with transcription

The expression of the thyroid-stimulating hormone (TSH) receptor and the cAMP-dependent protein kinase RII beta regulatory subunit confers TSH-cAMP-dependent growth to mouse fibroblasts.

TSH activates its specific receptor in thyroid cells and induces cAMP, a robust stimulator of thyroid cell proliferation. Conversely, cAMP is a potent inhibitor of growth in mouse fibroblasts. To dissect the signals mediating cAMP-dependent growth, we have expressed in mouse fibroblasts the human thyrotropin receptor (TSHR) or a constitutively active mutant, under the control of the tetracyclin promoter. Both TSHR and cAMP levels were modulated by tetracyclin. In the presence of serum, activation of TSHR by TSH induced growth arrest. In the absence of serum, cells expressing TSHR stimulated with TSH, replicated their DNA, but underwent apoptosis. Co-expression of cAMP-dependent protein kinase (PKA) regulatory subunit type II (RIIbeta) inhibited apoptosis and stimulated the growth of cells only in the presence of TSH. Expression of RIIbeta-PKA, in the absence of TSHR, induced apoptosis, which was reversed by cAMP. Growth, stimulated by TSHR-RIIbeta-PKA in mouse fibroblasts, was also dependent on Rap1 activity, indicating cAMP-dependent growth in thyroid cells. As for the molecular mechanism underlying these effects, we found that in normal fibroblasts, TSH induced AKT and ERK1/2 only in cells expressing TSHR and RII. Similarly, activation of TSHR increased cAMP levels greatly, but was unable to stimulate CREB phosphorylation and transcription of cAMP-induced genes in the absence of RII. These data provide a simple explanation for the anti-proliferative and proliferative effects of cAMP in different cell types and indicate that RII-PKAII complements TSHR action by stably propagating robust cAMP signals in cell compartments

Tracing and tracking epiallele families in complex DNA populations

DNA methylation is a stable epigenetic modification, extremely polymorphic and driven by stochastic and deterministic events. Most of the current techniques used to analyse methylated sequences identify methylated cytosines (mCpGs) at a single-nucleotide level and compute the average methylation of CpGs in the population of molecules. Stable epialleles, i.e. CpG strings with the same DNA sequence containing a discrete linear succession of phased methylated/non-methylated CpGs in the same DNA molecule, cannot be identified due to the heterogeneity of the 5′–3′ ends of the molecules. Moreover, these are diluted by random unstable methylated CpGs and escape detection. We present here MethCoresProfiler, an R-based tool that provides a simple method to extract and identify combinations of methylated phased CpGs shared by all components of epiallele families in complex DNA populations. The methylated cores are stable over time, evolve by acquiring or losing new methyl sites and, ultimately, display high information content and low stochasticity. We have validated this method by identifying and tracing rare epialleles and their families in synthetic or in vivo complex cell populations derived from mouse brain areas and cells during postnatal differentiation

RNA stabilizes transcription-Dependent Chromatin Loops Induced By Nuclear Hormones

We show that transcription induced by nuclear receptors for estrogen (e2) or retinoic acid (RA) is associated with formation of chromatin loops that juxtapose the 5’ end (containing the promoter) with the enhancer and the 3′ polyA addition site of the target gene. We nd three loop con gurations which change as a function of time after induction: 1. RA or E2-induced loops which connect the 5′ end, the enhancer and the 3′ end of the gene, and are stabilized by RNA early after induction; 2. E2-independent loops whose stability does not require RNA; 3. Loops detected only by treatment of chromatin with RNAse H1 prior to hormonal induction. RNAse H1 digests RNA that occludes the relevant restriction sites, thus preventing detection of these loops. R-loops at the 5′ and 3′ ends of the RA or e2-target genes were demonstrated by immunoprecipitation with anti-DNA-RNA hybrid antibodies as well as by sensitivity to RNAse H1. The cohesin RAD21 subunit is preferentially recruited to the target sites upon RA or e2 induction of transcription. R21 binding to chromatin is eliminated by RNAse H1. We identi ed e2-induced and RNase H1-sensitive antisense RNAs located at the 5′ and 3′ ends of the e2-induced transcription unit which stabilize the loops and RAD21 binding to chromatin. This is the rst report of chromatin loops that form after gene induction that are maintained by RNA:DNA hybrids

DNA damage and Repair Modify DNA methylation and Chromatin Domain of the Targeted Locus: Mechanism of allele methylation polymorphism

We characterize the changes in chromatin structure, DNA methylation and transcription during and after homologous DNA repair (HR). We find that HR modifies the DNA methylation pattern of the repaired segment. HR also alters local histone H3 methylation as well chromatin structure by inducing DNA-chromatin loops connecting the 5' and 3' ends of the repaired gene. During a two-week period after repair, transcription-associated demethylation promoted by Base Excision Repair enzymes further modifies methylation of the repaired DNA. Subsequently, the repaired genes display stable but diverse methylation profiles. These profiles govern the levels of expression in each clone. Our data argue that DNA methylation and chromatin remodelling induced by HR may be a source of permanent variation of gene expression in somatic cells