Roles for H2A.Z and Its Acetylation in GAL1 Transcription and Gene Induction, but Not GAL1-Transcriptional Memory

H2A.Z does not appear to have a role in GAL1 transcriptional memory, but it does have both acetylation-dependent and acetylation-independent roles in GAL1 induction and expression

The SWR1 Histone Replacement Complex Causes Genetic Instability and Genome-Wide Transcription Misregulation in the Absence of H2A.Z

The SWR1 complex replaces the canonical histone H2A with the variant H2A.Z (Htz1 in yeast) at specific chromatin regions. This dynamic alteration in nucleosome structure provides a molecular mechanism to regulate transcription, gene silencing, chromosome segregation and DNA repair. Here we show that genetic instability, sensitivity to drugs impairing different cellular processes and genome-wide transcriptional misregulation in htz1Δ can be partially or totally suppressed if SWR1 is not formed (swr1Δ), if it forms but cannot bind to chromatin (swc2Δ) or if it binds to chromatin but lacks histone replacement activity (swc5Δ and the ATPase-dead swr1-K727G). These results suggest that in htz1Δ the nucleosome remodelling activity of SWR1 affects chromatin integrity because of an attempt to replace H2A with Htz1 in the absence of the latter. This would impair transcription and, either directly or indirectly, other cellular processes. Specifically, we show that in htz1Δ, the SWR1 complex causes an accumulation of recombinogenic DNA damage by a mechanism dependent on phosphorylation of H2A at Ser129, a modification that occurs in response to DNA damage, suggesting that the SWR1 complex impairs the repair of spontaneous DNA damage in htz1Δ. In addition, SWR1 causes DSBs sensitivity in htz1Δ; consistently, in the absence of Htz1 the SWR1 complex bound near an endonuclease HO-induced DSB at the mating-type (MAT) locus impairs DSB-induced checkpoint activation. Our results support a stepwise mechanism for the replacement of H2A with Htz1 and demonstrate that a tight control of this mechanism is essential to regulate chromatin dynamics but also to prevent the deleterious consequences of an incomplete nucleosome remodelling

Genome-Wide Analysis of Factors Affecting Transcription Elongation and DNA Repair: A New Role for PAF and Ccr4-Not in Transcription-Coupled Repair

RNA polymerases frequently deal with a number of obstacles during transcription elongation that need to be removed for transcription resumption. One important type of hindrance consists of DNA lesions, which are removed by transcription-coupled repair (TC-NER), a specific sub-pathway of nucleotide excision repair. To improve our knowledge of transcription elongation and its coupling to TC-NER, we used the yeast library of non-essential knock-out mutations to screen for genes conferring resistance to the transcription-elongation inhibitor mycophenolic acid and the DNA-damaging agent 4-nitroquinoline-N-oxide. Our data provide evidence that subunits of the SAGA and Ccr4-Not complexes, Mediator, Bre1, Bur2, and Fun12 affect transcription elongation to different extents. Given the dependency of TC-NER on RNA Polymerase II transcription and the fact that the few proteins known to be involved in TC-NER are related to transcription, we performed an in-depth TC-NER analysis of a selection of mutants. We found that mutants of the PAF and Ccr4-Not complexes are impaired in TC-NER. This study provides evidence that PAF and Ccr4-Not are required for efficient TC-NER in yeast, unraveling a novel function for these transcription complexes and opening new perspectives for the understanding of TC-NER and its functional interconnection with transcription elongation

Dissection of Pol II Trigger Loop Function and Pol II Activity–Dependent Control of Start Site Selection In Vivo

Structural and biochemical studies have revealed the importance of a conserved, mobile domain of RNA Polymerase II (Pol II), the Trigger Loop (TL), in substrate selection and catalysis. The relative contributions of different residues within the TL to Pol II function and how Pol II activity defects correlate with gene expression alteration in vivo are unknown. Using Saccharomyces cerevisiae Pol II as a model, we uncover complex genetic relationships between mutated TL residues by combinatorial analysis of multiply substituted TL variants. We show that in vitro biochemical activity is highly predictive of in vivo transcription phenotypes, suggesting direct relationships between phenotypes and Pol II activity. Interestingly, while multiple TL residues function together to promote proper transcription, individual residues can be separated into distinct functional classes likely relevant to the TL mechanism. In vivo, Pol II activity defects disrupt regulation of the GTP-sensitive IMD2 gene, explaining sensitivities to GTP-production inhibitors, but contrasting with commonly cited models for this sensitivity in the literature. Our data provide support for an existing model whereby Pol II transcriptional activity provides a proxy for direct sensing of NTP levels in vivo leading to IMD2 activation. Finally, we connect Pol II activity to transcription start site selection in vivo, implicating the Pol II active site and transcription itself as a driver for start site scanning, contravening current models for this process

Polo kinase recruitment via the constitutive centromere-associated network at the kinetochore elevates centromeric RNA

The kinetochore, a multi-protein complex assembled on centromeres, is essential to segregate chromosomes during cell division. Deficiencies in kinetochore function can lead to chromosomal instability and aneuploidy-a hallmark of cancer cells. Kinetochore function is controlled by recruitment of regulatory proteins, many of which have been documented, however their function often remains uncharacterized and many are yet to be identified. To identify candidates of kinetochore regulation we used a proteome-wide protein association strategy in budding yeast and detected many proteins that are involved in post-translational modifications such as kinases, phosphatases and histone modifiers. We focused on the Polo-like kinase, Cdc5, and interrogated which cellular components were sensitive to constitutive Cdc5 localization. The kinetochore is particularly sensitive to constitutive Cdc5 kinase activity. Targeting Cdc5 to different kinetochore subcomplexes produced diverse phenotypes, consistent with multiple distinct functions at the kinetochore. We show that targeting Cdc5 to the inner kinetochore, the constitutive centromere-associated network (CCAN), increases the levels of centromeric RNA via an SPT4 dependent mechanism

Identification of a Novel Secretogranin II-Derived Peptide (SgII 187-252 ) in Adult and Fetal Human Adrenal Glands Using Antibodies Raised against the Human Recombinant Peptide*

International audienceMolecular cloning of secretogranin II (SgII) in phylogenetically distant species has recently revealed the existence of a highly conserved 66-amino acid peptide flanked by preserved pairs of basic residues. This observation suggested that this peptide, named EM66, which had not been described to date, could be an important processing product of SgII. The aim of the present study was to investigate the possible occurrence of EM66 in the human adrenal gland. The EM66 peptide was generated in Escherichia coli, which was programmed to make a fusion protein containing the human EM66 sequence. The affinity-purified fusion protein was used to raise poly-clonal antibodies in rabbits. The free EM66 peptide was obtained by cleavage of the fusion protein followed by high performance liquid chromatography purification. Immunohistochemical analysis using the EM66 antibodies revealed intense labeling of adrenochromaffin cells in the adult adrenal medulla and the fetal adrenal gland. A sensitive and specific RIA was developed and applied to the detection of EM66-like immunoreactivity in extracts of adult adrenal medulla and whole fetal adrenal gland after high performance liquid chro-matographic analysis. A major immunoreactive species exhibiting the same retention time as recombinant EM66 was detected in both adult and fetal adrenal extracts. Taken together, these data demonstrate that posttranslational processing of SgII actually generates EM66 in the adrenal gland. The strong conservation of the amino acid sequence of EM66 in the vertebrate phylum and the occurrence of the mature peptide in both fetal and adult chromaffin cells suggest that EM66 could play an important physiological role in the human adrenal gland. (J Clin Endocrinol Metab 83: 2944-2951, 1998

Identification of a Novel Secretogranin II-Derived Peptide (SgII 187-252 ) in Adult and Fetal Human Adrenal Glands Using Antibodies Raised against the Human Recombinant Peptide*

International audienceMolecular cloning of secretogranin II (SgII) in phylogenetically distant species has recently revealed the existence of a highly conserved 66-amino acid peptide flanked by preserved pairs of basic residues. This observation suggested that this peptide, named EM66, which had not been described to date, could be an important processing product of SgII. The aim of the present study was to investigate the possible occurrence of EM66 in the human adrenal gland. The EM66 peptide was generated in Escherichia coli, which was programmed to make a fusion protein containing the human EM66 sequence. The affinity-purified fusion protein was used to raise poly-clonal antibodies in rabbits. The free EM66 peptide was obtained by cleavage of the fusion protein followed by high performance liquid chromatography purification. Immunohistochemical analysis using the EM66 antibodies revealed intense labeling of adrenochromaffin cells in the adult adrenal medulla and the fetal adrenal gland. A sensitive and specific RIA was developed and applied to the detection of EM66-like immunoreactivity in extracts of adult adrenal medulla and whole fetal adrenal gland after high performance liquid chro-matographic analysis. A major immunoreactive species exhibiting the same retention time as recombinant EM66 was detected in both adult and fetal adrenal extracts. Taken together, these data demonstrate that posttranslational processing of SgII actually generates EM66 in the adrenal gland. The strong conservation of the amino acid sequence of EM66 in the vertebrate phylum and the occurrence of the mature peptide in both fetal and adult chromaffin cells suggest that EM66 could play an important physiological role in the human adrenal gland. (J Clin Endocrinol Metab 83: 2944-2951, 1998

Identification of a Novel Secretogranin II-Derived Peptide (SgII 187–252 ) in Adult and Fetal Human Adrenal Glands Using Antibodies Raised against the Human Recombinant Peptide*

International audienceMolecular cloning of secretogranin II (SgII) in phylogenetically distant species has recently revealed the existence of a highly conserved 66-amino acid peptide flanked by preserved pairs of basic residues. This observation suggested that this peptide, named EM66, which had not been described to date, could be an important processing product of SgII. The aim of the present study was to investigate the possible occurrence of EM66 in the human adrenal gland. The EM66 peptide was generated in Escherichia coli, which was programmed to make a fusion protein containing the human EM66 sequence. The affinity-purified fusion protein was used to raise poly-clonal antibodies in rabbits. The free EM66 peptide was obtained by cleavage of the fusion protein followed by high performance liquid chromatography purification. Immunohistochemical analysis using the EM66 antibodies revealed intense labeling of adrenochromaffin cells in the adult adrenal medulla and the fetal adrenal gland. A sensitive and specific RIA was developed and applied to the detection of EM66-like immunoreactivity in extracts of adult adrenal medulla and whole fetal adrenal gland after high performance liquid chro-matographic analysis. A major immunoreactive species exhibiting the same retention time as recombinant EM66 was detected in both adult and fetal adrenal extracts. Taken together, these data demonstrate that posttranslational processing of SgII actually generates EM66 in the adrenal gland. The strong conservation of the amino acid sequence of EM66 in the vertebrate phylum and the occurrence of the mature peptide in both fetal and adult chromaffin cells suggest that EM66 could play an important physiological role in the human adrena

Identification of a novel secretogranin II-derived peptide in the adult and fetal human adrenal gland.

International audienceMolecular cloning of secretogranin II (SgII) in different species has revealed the existence of a highly conserved 66-amino acid peptide (EM66) flanked by preserved pairs of basic residues. In the present study we have localized and characterized EM66 in the human adrenal gland. A fusion protein containing the human EM66 peptide was produced in E. coli and used to raise polyclonal antibodies in rabbits. Immunohistochemical staining of human adrenal slices revealed intense labeling of adrenochromaffin cells in the adult and fetal gland. HPLC analysis of adrenal extracts showed the presence of an immunoreactive peak exhibiting the same retention time as recombinant EM66 in both adult and fetus. These data demonstrate that post-translational processing of SgII actually generates a novel peptide in the human adrenal gland. The conservation of the sequence of EM66 in vertebrates and the occurrence of the mature peptide during early ontogenesis of the human adrenal gland strongly suggest that EM66 could exert physiological activities