CHD1 controls H3.3 incorporation in adult brain chromatin to maintain metabolic homeostasis and normal lifespan.

The ATP-dependent chromatin remodeling factor CHD1 is essential for the assembly of variant histone H3.3 into paternal chromatin during sperm chromatin remodeling in fertilized eggs. It remains unclear, however, if CHD1 has a similar role in normal diploid cells. Using a specifically tailored quantitative mass spectrometry approach, we show that Chd1 disruption results in reduced H3.3 levels in heads of Chd1 mutant flies. Chd1 deletion perturbs brain chromatin structure in a similar way as H3.3 deletion and leads to global de-repression of transcription. The physiological consequences are reduced food intake, metabolic alterations, and shortened lifespan. Notably, brain-specific CHD1 expression rescues these phenotypes. We further demonstrate a strong genetic interaction between Chd1 and H3.3 chaperone Hira. Thus, our findings establish CHD1 as a factor required for the assembly of H3.3-containing chromatin in adult cells and suggest a crucial role for CHD1 in the brain as a regulator of organismal health and longevity.

CHD1 contributes to intestinal resistance against infection by P. aeruginosa in Drosophila melanogaster.

Drosophila SNF2-type ATPase CHD1 catalyzes the assembly and remodeling of nucleosomal arrays in vitro and is involved in H3.3 incorporation in viin vivo during early embryo development. Evidence for a role as transcriptional regulator comes from its colocalization with elongating RNA polymerase II as well as from studies of fly Hsp70 transcription. Here we used microarray analysis to identify target genes of CHD1. We found a fraction of genes that were misregulated in Chd1 mutants to be functionally linked to Drosophila immune and stress response. Infection experiments using different microbial species revealed defects in host defense in Chd1-deficient adults upon oral infection with P. aeruginosa but not upon septic injury, suggesting a so far unrecognized role for CHD1 in intestinal immunity. Further molecular analysis showed that gut-specific transcription of antimicrobial peptide genes was overactivated in the absence of infection in Chd1 mutant flies. Moreover, microbial colonization of the intestine was elevated in Chd1 mutants and oral infection resulted in strong enrichment of bacteria in the body cavity indicating increased microbial passage across intestinal epithelia. However, we did not detect enhanced epithelial damage or alterations of the intestinal stem cell population. Collectively, our data provide evidence that intestinal resistance against infection by P. aeruginosa in Drosophila is linked to maintaining proper balance of gut-microbe interactions and that the chromatin remodeler CHD1 is involved in regulating this aspect.

The chromodomains of CHD1 are critical for enzymatic activity but less important for chromatin localization.

The molecular motor protein CHD1 has been implicated in the regulation of transcription and in the transcription-independent genome-wide incorporation of H3.3 into paternal chromatin in Drosophila melanogaster. A key feature of CHD1 is the presence of two chromodomains, which can bind to histone H3 methylated at lysine 4 and thus might serve to recruit and/or maintain CHD1 at the chromatin. Here, we describe genetic and biochemical approaches to the study of the Drosophila CHD1 chromodomains. We found that overall localization of CHD1 on polytene chromosomes does not appreciably change in chromodomain-mutant flies. In contrast, the chromodomains are important for transcription-independent activities of CHD1 during early embryonic development as well as for transcriptional regulation of several heat shock genes. However, neither CHD1 nor its chromodomains are needed for RNA polymerase II localization and H3K4 methylation but loss of CHD1 decreases transcription-induced histone eviction at the Hsp70 gene in vivo. Chromodomain mutations negatively affect the chromatin assembly activities of CHD1 in vitro, and they appear to be involved in linking the ATP-dependent motor to the chromatin assembly function of CHD1.

CenH3/CID incorporation is not dependent on the chromatin assembly factor CHD1 in Drosophila.

CHD1 is a SNF2-related ATPase that is required for the genome-wide incorporation of variant histone H3.3 in the paternal pronucleus as well as in transcriptionally active nuclei in Drosophila embryos. The S. pombe and vertebrate orthologs of CHD1 have been implicated in the assembly of the centromeric histone H3 variant CenH3(CENP-A), which occurs in a DNA replication-independent manner. Here, we examined whether CHD1 participates in the assembly of CenH3(CID) in Drosophila. In contrast to the findings in fission yeast and vertebrate cells, our evidence clearly argues against such a role for CHD1 in Drosophila. CHD1 does not localize to centromeres in either S2 cells or developing fly embryos. Down-regulation of CHD1 in S2 cells by RNAi reveals unchanged levels of CenH3(CID) at the centromeres. Most notably, ablation of functional CHD1 in Chd1 mutant fly embryos does not interfere with centromere and kinetochore assembly, as the levels and localization of CenH3(CID), CENP-C and BubR1 in the mutant embryos remain similar to those seen in wild-type embryos. These results indicate that Drosophila CHD1 has no direct function in the incorporation of the centromeric H3 variant CenH3(CID) into chromatin. Therefore, centromeric chromatin assembly may involve different mechanisms in different organisms.

p42/44 MAPK is an essential effector for purine nucleoside-mediated neuroprotection of hypoxic PC12 cells and primary cerebellar granule neurons.

Purine nucleosides protect neurons against hypoxic insult, but the signaling mechanisms have not yet been fully elucidated. We studied the role of the p42/44 MAPK pathway in purine nucleoside-mediated protection of cultured PC12 cells and primary cerebellar granule neurons from hypoxia-induced cell death. Incubation with adenosine reduced hypoxia-evoked cell death morphology, and increased the activity of the MAPK pathway. Inosine, a metabolic derivative of adenosine was generally less potent in PC12 cells. Pharmacological inhibition of the MAPK pathways severely hampered adenosine-mediated induction of cell viability and neurite outgrowth. Consistently, siRNA-mediated knockdown of p42 and p44 MAPK completely blocked adenosine-mediated rescue of hypoxic PC12 cells. The role of MAPK activation was further studied in primary neurons. Cells were significantly rescued by adenosine and inosine and siRNA-mediated knockdown severely affected purine-mediated rescue of neuronal viability after hypoxic insult. Results point to the important role of p42/44 MAPK for adenosine receptor-mediated neuroprotection.

ATP-dependent chromatin remodeling enzymes and their various roles in cell cycle control.

The modification of chromatin structure by various mechanisms has emerged as a key regulatory component of nuclear programs. Cell cycle progression and exit are affected by the integrity of chromatin architecture as well as by regulatory cues that chromatin structure imposes on the expression of cell cycle genes. ATP-dependent chromatin remodeling factors use the energy derived from ATP-hydrolysis to modulate histone-DNA contacts. These molecular machines play important roles in all aspects of chromosome biology and are thus intimately linked to cell cycle control. Regulation of complex activity by various signaling pathways has been a rising theme in recent years. Moreover, some chromatin remodeling factors have been characterized as potent tumor suppressor proteins. Thus, to understand the functions and activities of ATP-utilizing chromatin remodeling factors is an important goal towards their use as potential targets in cancer therapy.

CHD1 motor protein is required for deposition of histone variant H3.3 into chromatin in vivo.

The organization of chromatin affects all aspects of nuclear DNA metabolism in eukaryotes. H3.3 is an evolutionarily conserved histone variant and a key substrate for replication-independent chromatin assembly. Elimination of chromatin remodeling factor CHD1 in Drosophila embryos abolishes incorporation of H3.3 into the male pronucleus, renders the paternal genome unable to participate in zygotic mitoses, and leads to the development of haploid embryos. Furthermore, CHD1, but not ISWI, interacts with HIRA in cytoplasmic extracts. Our findings establish CHD1 as a major factor in replacement histone metabolism in the nucleus and reveal a critical role for CHD1 in the earliest developmental instances of genome-scale, replication-independent nucleosome assembly. Furthermore, our results point to the general requirement of adenosine triphosphate (ATP)-utilizing motor proteins for histone deposition in vivo.

Purine nucleoside-mediated protection of chemical hypoxia-induced neuronal injuries involves p42/44 MAPK activation.

Hypoxia in brain may lead to cell death by apoptosis and necrosis. Concomitant is the formation of purine nucleosides, e.g. adenosine, a powerful endogenous neuroprotectant. Despite vigorous studies, many aspects of the mechanisms involved in purine-based protection are still unclear. In this study, we wanted to investigate the effect of purine nucleosides on cellular responses to chemical hypoxia. O(2)-sensitive neuronal pheochromocytoma (PC12)-cells, which are widely used as a model system for sympathetic ganglion-like neurons, were subjected to chemical hypoxia induced with rotenone, an inhibitor of mitochondrial complex I. Adenosine and its relatives guanosine and inosine were tested for their neuroprotective capability to improve neurite outgrowth and viability. In addition, cell lysates were analyzed for mitogen-activated-protein-kinases (MAPK) activation by anti-active and anti-total MAPKinase immunoblotting. Adenosine, guanosine and inosine significantly inhibited the loss of viability after hypoxic insult. In combination with NGF, purine nucleosides also partially rescued neurite outgrowth. The MEK-1/-2 inhibitor PD098059 inhibited purine nucleoside-mediated protection up to 85.23% and also markedly decreased neurite formation induced by NGF and purine nucleosides in hypoxic cells. Immunoblot analysis revealed a strong activation of MAPKinase upon incubation of cells with adenosine, guanosine or inosine. In combination with NGF an additive effect was observed. Results suggested that activation of the MAPKinase pathway plays a vital role in purine nucleoside-mediated protection of neuronal cells following hypoxic insult.

Purine nucleosides support the neurite outgrowth of primary rat cerebellar granule cells after hypoxia.

Mammalian neurons require a constant supply of oxygen to maintain adequate cellular functions and survival. Following sustained hypoxia during ischemic events in brain, the energy status of neurons and glia is compromised, which may subsequently lead to cell death by apoptosis and necrosis. Concomitant with energy depletion is the formation of the purine nucleoside adenosine, a powerful endogenous neuroprotectant. In this paper the effect of chemical hypoxia on cell survival and neurite outgrowth of primary cerebellar granule cells was investigated. Rotenone, a mitochondrial complex I inhibitor, induced a 30.4 +/- 3.6% loss of viable cells and a 35.0 +/- 4.4% loss of neurite formation of cerebellar granule cells, which was partially restored by the addition of purine nucleosides adenosine, inosine and guanosine. Inosine had the most striking effect of 37.7 +/- 2.9% rescue of viability and 71.2 +/- 18.4% rescue of neurite outgrowth. Data confirm the suggested role of purine nucleosides for the neuronal regeneration of primary brain cells following hypoxic insult.