Mustapha Oulad-Abdelghani

Heterochromatin Formation in Mammalian Cells: Interaction between Histones and HP1 Proteins

Members of the heterochromatin protein 1 (HP1) family are silencing nonhistone proteins. Here, we show that in P19 embryonal carcinoma (EC) nuclei, HP1 alpha, beta, and gamma form homo- and heteromers associated with nucleosomal core histones. In vitro, all three HP1s bind to tailed and tailless nucleosomes and specifically interact with the histone-fold of histone H3. Furthermore, HP1alpha interacts with the linker histone H1. HP1alpha binds to H3 and H1 through its chromodomain (CD) and hinge region, respectively. Interestingly, the Polycomb (Pc1/M33) CD also interacts with H3, and HP1alpha and Pc1/M33 binding to H3 is severely impaired by CD mutations known to abrogate HP1 and Polycomb silencing in Drosophila. These results define a novel function for the conserved CD and suggest that HP1 self-association and histone binding may play a crucial role in HP1-mediated heterochromatin assembly.

Interaction with members of the heterochromatin protein 1 (HP1) family and histone deacetylation are differentially involved in transcriptional silencing by members of the TIF1 family

Mammalian TIF1α and TIF1β (KAP‐1/KRIP‐1) are related transcriptional intermediary factors that possess intrinsic silencing activity. TIF1α is believed to be a euchromatic target for liganded nuclear receptors, while TIF1β may serve as a co‐repressor for the large family of KRAB domain‐containing zinc finger proteins. Here, we report an association of TIF1β with both heterochromatin and euchromatin in interphase nuclei. Co‐immunoprecipitation of nuclear extracts shows that endogenous TIF1β, but not TIF1α, is associated with members of the heterochromatin protein 1 (HP1) family. However, in vitro, both TIF1α and TIF1β interact with and phosphorylate the HP1 proteins. This interaction involves a conserved amino acid motif, which is critical for the silencing activity of TIF1β but not TIF1α. We further show that trichostatin A, an inhibitor of histone deacetylases, can interfere with both TIF1 and HP1 silencing. The silencing activity of TIF1α appears to result chiefly from histone deacetylation, whereas that of TIF1β may be mediated via both HP1 binding and histone deacetylation.

Nodal Antagonists in the Anterior Visceral Endoderm Prevent the Formation of Multiple Primitive Streaks

The anterior visceral endoderm plays a pivotal role in establishing anterior-posterior polarity of the mouse embryo, but the molecular nature of the signals required remains to be determined. Here, we demonstrate that Cerberus-like(-/-);Lefty1(-/-) compound mutants can develop a primitive streak ectopically in the embryo. This defect is not rescued in chimeras containing wild-type embryonic, and Cerberus-like(-/-);Lefty1(-/-) extraembryonic, cells but is rescued in Cerberus-like(-/-); Lefty1(-/-) embryos after removal of one copy of the Nodal gene. Our findings provide support for a model whereby Cerberus-like and Lefty1 in the anterior visceral endoderm restrict primitive streak formation to the posterior end of mouse embryos by antagonizing Nodal signaling. Both antagonists are also required for proper patterning of the primitive streak.

H3K9 and H3K14 acetylation co-occur at many gene regulatory elements, while H3K14ac marks a subset of inactive inducible promoters in mouse embryonic stem cells

BackgroundTranscription regulation in pluripotent embryonic stem (ES) cells is a complex process that involves multitude of regulatory layers, one of which is post-translational modification of histones. Acetylation of specific lysine residues of histones plays a key role in regulating gene expression.ResultsHere we have investigated the genome-wide occurrence of two histone marks, acetylation of histone H3K9 and K14 (H3K9ac and H3K14ac), in mouse embryonic stem (mES) cells. Genome-wide H3K9ac and H3K14ac show very high correlation between each other as well as with other histone marks (such as H3K4me3) suggesting a coordinated regulation of active histone marks. Moreover, the levels of H3K9ac and H3K14ac directly correlate with the CpG content of the promoters attesting the importance of sequences underlying the specifically modified nucleosomes. Our data provide evidence that H3K9ac and H3K14ac are also present over the previously described bivalent promoters, along with H3K4me3 and H3K27me3. Furthermore, like H3K27ac, H3K9ac and H3K14ac can also differentiate active enhancers from inactive ones. Although, H3K9ac and H3K14ac, a hallmark of gene activation exhibit remarkable correlation over active and bivalent promoters as well as distal regulatory elements, a subset of inactive promoters is selectively enriched for H3K14ac.ConclusionsOur study suggests that chromatin modifications, such as H3K9ac and H3K14ac, are part of the active promoter state, are present over bivalent promoters and active enhancers and that the extent of H3K9 and H3K14 acetylation could be driven by cis regulatory elements such as CpG content at promoters. Our study also suggests that a subset of inactive promoters is selectively and specifically enriched for H3K14ac. This observation suggests that histone acetyl transferases (HATs) prime inactive genes by H3K14ac for stimuli dependent activation. In conclusion our study demonstrates a wider role for H3K9ac and H3K14ac in gene regulation than originally thought.

UV‐damaged DNA‐binding protein in the TFTC complex links DNA damage recognition to nucleosome acetylation

Initiation of transcription of protein‐encoding genes by RNA polymerase II (Pol II) was thought to require transcription factor TFIID, a complex comprised of the TATA box‐binding protein (TBP) and TBP‐associated factors (TAFIIs). In the presence of TBP‐free TAFII complex (TFTC), initiation of Pol II transcription can occur in the absence of TFIID. TFTC containing the GCN5 acetyltransferase acetylates histone H3 in a nucleosomal context. We have identified a 130 kDa subunit of TFTC (SAP130) that shares homology with the large subunit of UV‐damaged DNA‐binding factor. TFTC preferentially binds UV‐irradiated DNA, UV‐damaged DNA inhibits TFTC‐mediated Pol II transcription and TFTC is recruited in parallel with the nucleotide excision repair protein XP‐A to UV‐damaged DNA. TFTC preferentially acetylates histone H3 in nucleosomes assembled on UV‐damaged DNA. In agreement with this, strong histone H3 acetylation occurs in intact cells after UV irradiation. These results suggest that the access of DNA repair machinery to lesions within chromatin may be facilitated by TFTC via covalent modification of chromatin. Thus, our experiments reveal a molecular link between DNA damage recognition and chromatin modification.

STRA8-deficient spermatocytes initiate, but fail to complete, meiosis and undergo premature chromosome condensation

We analysed the phenotypic outcome of a Stra8-null mutation on male meiosis. Because the mutant spermatocytes (1) underwent premeiotic DNA replication, (2) displayed cytological features attesting initiation of recombination and of axial-element assembly, and (3) expressed Spo11 and numerous other meiotic genes, it was concluded that STRA8 is dispensable for meiotic initiation. The few mutant spermatocytes that progressed beyond leptonema showed a prolonged bouquet-stage configuration, asynapsis and heterosynapsis, suggesting function(s) of STRA8 in chromosome pairing. Most importantly, a large number of mutant leptotene spermatocytes underwent premature chromosome condensation, within 24 hours following the meiotic S phase. This phenomenon yielded aberrant metaphase-like cells with 40 univalent chromosomes, similar to normal mitotic metaphases. From these latter observations and from the wild-type pattern of Stra8 expression, we propose that, in preleptotene spermatocytes, STRA8 is involved in the process that leads to stable commitment to the meiotic cell cycle.

Loss of C9ORF72 impairs autophagy and synergizes with polyQ Ataxin‐2 to induce motor neuron dysfunction and cell death

An intronic expansion of GGGGCC repeats within the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (ALS‐FTD). Ataxin‐2 with intermediate length of polyglutamine expansions (Ataxin‐2 Q30x) is a genetic modifier of the disease. Here, we found that C9ORF72 forms a complex with the WDR41 and SMCR8 proteins to act as a GDP/GTP exchange factor for RAB8a and RAB39b and to thereby control autophagic flux. Depletion of C9orf72 in neurons partly impairs autophagy and leads to accumulation of aggregates of TDP‐43 and P62 proteins, which are histopathological hallmarks of ALS‐FTD. SMCR8 is phosphorylated by TBK1 and depletion of TBK1 can be rescued by phosphomimetic mutants of SMCR8 or by constitutively active RAB39b, suggesting that TBK1, SMCR8, C9ORF72, and RAB39b belong to a common pathway regulating autophagy. While depletion of C9ORF72 only has a partial deleterious effect on neuron survival, it synergizes with Ataxin‐2 Q30x toxicity to induce motor neuron dysfunction and neuronal cell death. These results indicate that partial loss of function of C9ORF72 is not deleterious by itself but synergizes with Ataxin‐2 toxicity, suggesting a double‐hit pathological mechanism in ALS‐FTD.

A new mouse member of the Wnt gene family, mWnt-8, is expressed during early embryogenesis and is ectopically induced by retinoic acid.

We have identified a novel mouse Wnt genc using a cDNA differential screening procedure for retinoic-acid-induced transcripts in P19 embryonal carcinoma cells. Sequence analysis showed that this gene represents the first murine Wnt-8 (mWnt-8) gene reported to date. The expression of the mWnt-8 gene, which is rapidly induced by retinoic acid in P19 and embryonic stem cells, appears to be restricted to early stages of mouse embryogenesis. mWnt-8 transcripts are first detected in the posterior region of the epiblast of early primitive streak-stage embryos. As gastrulation proceeds, mWnt-8 expression spreads into the embryonic ectoderm up to a sharp rostral boundary at the base of the developing headfolds. mWnt-8 is also transiently expressed in the newly formed mesoderm. mWnt-8 expression is rapidly down-regulated during early somitogenesis, the latest detectable expression domains corresponding to the presumptive fourth rhombomere and the caudal region of the neural plate. The expression pattern of mWnt-8 is clearly distinct from those of other murine Wnt genes expressed during gastrulation, but shows striking similarities with that of the chicken Cwnt-8C gene. We also show that mWnt-8 expression is ectopically induced in the rostral neural plate in response to RA exposure of presumitic (7-7.5 days post coitum) cultured mouse embryos.

MEIS2, A NOVEL MOUSE PBX-RELATED HOMEOBOX GENE INDUCED BY RETINOIC ACID DURING DIFFERENTIATION OF P19 EMBRYONAL CARCINOMA CELLS

We report the cDNA cloning, partial genomic organization, and expression pattern of Stra10, a novel retinoic acid‐inducible gene in P19 embryonal carcinoma cells. Four murine cDNA isoforms have been isolated, which are likely to result from alternative splicing. The predicted protein sequences exhibit approximately 85% identity with the Pbx‐related Meis1 homeobox gene products, which are involved in myeloid leukemia in BXH‐2 mice, and one of the Stra10 isoforms corresponds to the recently published Meis2 sequence (Nakamura et al. [1996] Oncogene 13:2235–2242). The Meis2 homeodomain is identical to that of Meis1, and is most closely related to those of the Pbx/TGIF homeobox gene products. By in situ hybridization analysis, we show that the Meis2 gene displays spatially restricted expression patterns in the developing nervous system, limbs, face, and in various viscera. In adult mice, Meis2 is mainly expressed in the brain and female genital tract, with a different distribution of the alternative splice forms in these organs. Dev. Dyn. 1997;210:173–183.

The Major Yolk Protein Vitellogenin Interferes with the Anti-Plasmodium Response in the Malaria Mosquito Anopheles gambiae

Functional gene analysis in malaria mosquitoes reveals molecules underpinning the trade-off between efficient reproduction and the antiparasitic response.