The Chromatin Accessibility Complex (CHRAC) consists of the ATPase ISWI, the large ACF1 subunit and a pair of small histone-like proteins, CHRAC-14/16. CHRAC is a prototypical nucleosome sliding factor that mobilizes nucleosomes to improve the regularity and integrity of the chromatin fiber. This may facilitate the formation of repressive chromatin. Expression of the signature subunit ACF1 is restricted during embryonic development, but remains high in primordial germ cells. Therefore, we explored roles for ACF1 during Drosophila oogenesis. ACF1 is expressed in somatic and germline cells, with notable enrichment in germline stem cells and oocytes. The asymmetrical localization of ACF1 to these cells depends on the transport of the Acf1 mRNA by the Bicaudal-D/Egalitarian complex. Loss of ACF1 function in the novel Acf17 allele leads to defective egg chambers and their elimination through apoptosis. In addition, we find a variety of unusual 16-cell cyst packaging phenotypes in the previously known Acf11 allele, with a striking prevalence of egg chambers with two functional oocytes at opposite poles. Surprisingly, we found that the Acf11 deletion – despite disruption of the Acf1 reading frame – expresses low levels of a PHD-bromodomain module from the C-terminus of ACF1 that becomes enriched in oocytes. Expression of this module from the Acf1 genomic locus leads to packaging defects in the absence of functional ACF1, suggesting competitive interactions with unknown target molecules. Remarkably, a two-fold overexpression of CHRAC (ACF1 and CHRAC-16) leads to increased apoptosis and packaging defects. Evidently, finely tuned CHRAC levels are required for proper oogenesis.

Malignant transformation is associated with loss of cell differentiation, anaplasia. Transcription factors gli, required for embryonic development, may be involved in this process. We studied the activity of transcription factors gli in high-grade gliomas and their role in maintenance of stem cell state and glioma cell survival. 20 glioma cell lines and a sample of a normal adult brain tissue were used in the present study. We found the expression of gli target genes, including GLI1 and FOXM1, in all tested glioma cell lines, but not in the normal tissue. Interestingly, the expression of gli target genes in some glioma cell lines was observed together with a high level of their transcriptional repressor, Gli3R. Knockdown of GLI3 in one of these lines resulted in decrease of gli target gene expression. These data suggest that Gli3R does not prevent the gli target genes transcription, and gli3 acts in glioma cells more as an activator, than a repressor of transcription. We observed that gli regulated the expression of such genes, as SOX2 or OCT4 that maintain stem cell state, and TET1, involving in DNA demethylation. Treatment with GANT61 or siRNA against GLI1, GLI2, or GLI3 could result in complete glioma cell death, while cyclopamine had a weaker and line-specific effect on glioma cell survival. Thus, the gli transcription factors are abnormally active in high-grade gliomas, regulate expression of genes, maintaining the stem cell state, and contribute to glioma cell survival.

Chromatin assembly is a fundamental process essential for chromosome duplication subsequent to DNA replication. In addition, histone removal and incorporation take place constantly throughout the cell cycle in the course of DNA-utilizing processes, such as transcription, damage repair or recombination. In vitro studies have revealed that nucleosome assembly relies on the combined action of core histone chaperones and ATP-utilizing molecular motor proteins such as ACF or CHD1. Despite extensive biochemical characterization of ATP-dependent chromatin assembly and remodeling factors, it has remained unclear to what extent nucleosome assembly is an ATP-dependent process in vivo. Our original and published data about the functions of ATP-dependent chromatin assembly and remodeling factors clearly demonstrated that these proteins are important for nucleosome assembly and histone exchange in vivo. During male pronucleus reorganization after fertilization CHD1 has a critical role in the genomescale, replication-independent nucleosome assembly involving the histone variant H3.3. Thus, the molecular motor proteins, such as CHD1, function not only in the remodeling of existing nucleosomes but also in de novo nucleosome assembly from DNA and histones in vivo. ATP-dependent chromatin assembly and remodeling factors have been implicated in the process of histone exchange during transcription and DNA repair, in the maintenance of centromeric chromatin and in the loading and remodeling of nucleosomes behind a replication fork. Thus, chromatin remodeling factors are involved in the processes of both replication-dependent and replication-independent chromatin assembly. The role of these proteins is especially prominent in the processes of large-scale chromatin reorganization; for example, during male pronucleus formation or in DNA repair. Together, ATP-dependent chromatin assembly factors, histone chaperones and chromatin modifying enzymes form a “chromatin integrity network” to ensure proper maintenance and propagation of chromatin landscape.

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