Anna Sala

The Nucleosome-Remodeling ATPase ISWI is Regulated by poly-ADP-ribosylation.

ATP-dependent nucleosome-remodeling enzymes and covalent modifiers of chromatin set the functional state of chromatin. However, how these enzymatic activities are coordinated in the nucleus is largely unknown. We found that the evolutionary conserved nucleosome-remodeling ATPase ISWI and the poly-ADP-ribose polymerase PARP genetically interact. We present evidence showing that ISWI is target of poly-ADP-ribosylation. Poly-ADP-ribosylation counteracts ISWI function in vitro and in vivo. Our work suggests that ISWI is a physiological target of PARP and that poly-ADP-ribosylation can be a new, important post-translational modification regulating the activity of ATP-dependent nucleosome remodelers.

Genome-wide characterization of chromatin binding and nucleosome spacing activity of the nucleosome remodelling ATPase ISWI

The evolutionarily conserved ATP‐dependent nucleosome remodelling factor ISWI can space nucleosomes affecting a variety of nuclear processes. In Drosophila, loss of ISWI leads to global transcriptional defects and to dramatic alterations in higher‐order chromatin structure, especially on the male X chromosome. In order to understand if chromatin condensation and gene expression defects, observed in ISWI mutants, are directly correlated with ISWI nucleosome spacing activity, we conducted a genome‐wide survey of ISWI binding and nucleosome positioning in wild‐type and ISWI mutant chromatin. Our analysis revealed that ISWI binds both genic and intergenic regions. Remarkably, we found that ISWI binds genes near their promoters causing specific alterations in nucleosome positioning at the level of the Transcription Start Site, providing an important insights in understanding ISWI role in higher eukaryote transcriptional regulation. Interestingly, differences in nucleosome spacing, between wild‐type and ISWI mutant chromatin, tend to accumulate on the X chromosome for all ISWI‐bound genes analysed. Our study shows how in higher eukaryotes the activity of the evolutionarily conserved nucleosome remodelling factor ISWI regulates gene expression and chromosome organization genome‐wide.

Genetic Identification of a Network of Factors that Functionally Interact with the Nucleosome Remodeling ATPase ISWI

Nucleosome remodeling and covalent modifications of histones play fundamental roles in chromatin structure and function. However, much remains to be learned about how the action of ATP-dependent chromatin remodeling factors and histone-modifying enzymes is coordinated to modulate chromatin organization and transcription. The evolutionarily conserved ATP-dependent chromatin-remodeling factor ISWI plays essential roles in chromosome organization, DNA replication, and transcription regulation. To gain insight into regulation and mechanism of action of ISWI, we conducted an unbiased genetic screen to identify factors with which it interacts in vivo. We found that ISWI interacts with a network of factors that escaped detection in previous biochemical analyses, including the Sin3A gene. The Sin3A protein and the histone deacetylase Rpd3 are part of a conserved histone deacetylase complex involved in transcriptional repression. ISWI and the Sin3A/Rpd3 complex co-localize at specific chromosome domains. Loss of ISWI activity causes a reduction in the binding of the Sin3A/Rpd3 complex to chromatin. Biochemical analysis showed that the ISWI physically interacts with the histone deacetylase activity of the Sin3A/Rpd3 complex. Consistent with these findings, the acetylation of histone H4 is altered when ISWI activity is perturbed in vivo. These findings suggest that ISWI associates with the Sin3A/Rpd3 complex to support its function in vivo.

Hsp10 nuclear localization and changes in lung cells response to cigarette smoke suggest novel roles for this chaperonin

Heat-shock protein (Hsp)10 is the co-chaperone for Hsp60 inside mitochondria, but it also resides outside the organelle. Variations in its levels and intracellular distribution have been documented in pathological conditions, e.g. cancer and chronic obstructive pulmonary disease (COPD). Here, we show that Hsp10 in COPD undergoes changes at the molecular and subcellular levels in bronchial cells from human specimens and derived cell lines, intact or subjected to stress induced by cigarette smoke extract (CSE). Noteworthy findings are: (i) Hsp10 occurred in nuclei of epithelial and lamina propria cells of bronchial mucosa from non-smokers and smokers; (ii) human bronchial epithelial (16HBE) and lung fibroblast (HFL-1) cells, in vitro, showed Hsp10 in the nucleus, before and after CSE exposure; (iii) CSE stimulation did not increase the levels of Hsp10 but did elicit qualitative changes as indicated by molecular weight and isoelectric point shifts; and (iv) Hsp10 nuclear levels increased after CSE stimulation in HFL-1, indicating cytosol to nucleus migration, and although Hsp10 did not bind DNA, it bound a DNA-associated protein.

Poly-ADP-Ribose (PAR) as an epigenetic flag

Epigenetics is the study of hereditable chromatin modifications, such as DNA methylation, histone modifications, and nucleosome-remodelling, which occur without alterations to the DNA sequence. The establishment of different epigenetic states in eukaryotes depends on regulatory mechanisms that induce structural changes in chromatin in response to environmental and cellular cues. Two classes of enzymes modulate chromatin accessibility: chromatin-covalent modifiers and ATP-dependent chromatin remodelling complexes. The first class of enzymes catalyzes covalent modifications of DNA as well as the amino- and carboxy-terminal tails of histones, while the second uses the energy of ATP hydrolysis to reposition nucleosomes along the chromatin fibers or to incorporate histone variants. Thus, epigenetic modifications are reversible nuclear reactions. In the last decade, many studies have strongly indicated that alterations in epigenetic modifications may contribute to the onset and progression of a variety of human diseases such as cancer. Therefore, the enzymes responsible for these chromatin changes are becoming attractive therapeutic targets.

RNA-binding activity of the rat calmodulin-binding PEP-19 protein and of the long PEP-19 isoform

Synthesis of H1˚ histone protein, in the developing rat brain, seems to be regulated mainly at the post-transcriptional level. Since regulation of RNA metabolism depends on a series of RNA-binding proteins, we have been searching for RNA-binding proteins involved in the post-transcriptional regulation of the H1˚ gene. We recently reported isolation, from a cDNA expression library, of an insert encoding a novel protein, the C-terminal half of which is identical to that of PEP-19, a brain-specific protein involved in calcium metabolism. The novel protein was called long PEP-19 isoform (LPI). Herein we show that LPI, as well as PEP-19, can bind H1˚ RNA. Moreover, in order to improve production of functional LPI/PEP-19, we modified the protocol normally adopted for preparing histidine tagged-proteins from bacteria, by adding an additional purification step. We also found that both LPI and PEP can compete for H1˚ RNA binding with PIPPin (CSD-C2), another RNA-binding protein previously discovered in our laboratory. Since PEP19/LPI contain a calmodulin binding domain, we finally investigated whether their ability to bind RNA is affected by calmodulin. Our results show that calmodulin interferes with binding of H1˚ RNA to both PEP-19 and LPI, while it is not able to bind RNA on its own. This finding suggests that calcium/calmodulin may have a role in controlling H1˚ mRNA metabolism in the developing brain.

Immunolocalization of Poly ADP-Ribose on Drosophila Polytene Chromosomes

Poly ADP-ribosylation (PARylation) is a posttranslational protein modification catalyzed by poly -ADP-ribose polymerases (PARPs). Poly ADP-ribose metabolism is involved in a wide range of biological processes, such as maintenance of genome stability, transcriptional regulation, energy metabolism, and programed cell death. Recently, chromatin components, including histones, have been shown to be targets of PARylation. Unlike mammals, which have several PARP-encoded genes, the model organism Drosophila melanogaster has only one PARP gene, highly related to mammalian PARP1. These features make flies a great model system to study PARP biology. Commercially available antibodies recognizing this covalent modification have made possible the development of immunofluorescence approaches to study PARylation of chromatin components. Here, we present a protocol to immunostain polytene chromosomes of the model system D. melanogaster.