Project 3. Histone Variants in Rodent Depression Models

(David Allis, Rockefeller; Ian Maze, Mount Sinai)

 

Exciting new research indicates that, in addition to post-translational modifications of histones, eukaryotic cells further generate chromatin structural variation through the introduction of variant histone proteins into existing nucleosomes. Emerging evidence from the Allis laboratory indicates that H3.3, a variant of histone H3, represents a unique chromatin mark, indicative of dynamic changes in gene expression that occur both early in life and in the fully differentiated adult brain. Importantly, H3.3 deposition is enriched in neurons and such enrichment grows during development, which illustrates the profound restructuring of chromatin that occurs in brain throughout an individual’s lifetime. Project 3 focuses on the role of this highly novel mode of chromatin regulation in rodent depression models, with parallel studies of human postmortem brain in Project 4. We have found that H3.3 expression increases in response to neural activity, induced optogenetically, or other stimuli in cultured mouse cortical neurons (Figure left), and have used ChIP-Seq to characterize, genome-wide, the specific genomic loci that exhibit altered H3.3 deposition. Early evidence identifies numerous genes implicated in neural plasticity, including several of interest to other Projects in this Center, such as CREB pathways. Meanwhile, we have found that optogenetic stimulation of mouse PFC neurons in vivo also induces total expression levels of H3.3 (Figure left). H3.3 expression in PFC is similarly induced after chronic social defeat stress (CSDS) in susceptible but not resilient animals (Figure right), while mild early life stress in the maternal separation stress model poises H3.3 for induction in NAc by CSDS (Figure right). Finally, we have detected opposite abnormal expression patterns of H3.3 in the PFC and NAc of depressed humans, with evidence for antidepressants normalizing these changes. We are now extending these highly original findings in the following ways. 1) We are identifying genome-wide patterns of histone H3.3 localization in PFC and NAc in response to CSDS and maternal separation stress (MS) using ChIP-Seq with highly specific antibodies generated and validated by our group. Such patterns are being compared to standard forms of H3, i.e., H3.1/2. Lists of H3.3-regulated loci are being compared with those induced upon optogenetic stimulation of PFC or NAc. These studies will thus enable us to link stress-induced changes with those achieved with direct manipulation of neural activity, possibly informing the therapeutic mechanisms of deep brain stimulation in depression noted above (11,66). 2) We are examining protein complexes containing H3.3, both basally and in response to CSDS. Such studies are possible since we have generated knockin mice where H3.3 or H3.1/2 is tagged. This work, in concert with the genome-wide assessments of standard histone modifications and other regulatory proteins in the Chromatin and Gene Analysis Core and in Projects 1 and 2, are helping us understand how H3.3 affects gene regulation in depression models. 3) We are determining the functional relevance of H3.3 in PFC and NAc after CSDS or MS by means of loss- and gain-of-function studies using novel tools, including conditional H3.3 knockout mice and viral vectors. 4) Such studies in the CSDS and MS models will be complemented, as time and resources allow, by examining other stress models. This work will thus provide fundamentally new information regarding the epigenome in depression models and help elaborate the pathophysiology of depression and pinpoint novel targets for therapeutic intervention. In this way, studies outlined in Project 3 provide a template for investigation of other histone variants, and of novel readers for these variants, in depression models.

Optogenetic Induction of H3.3 - Regulation of H3.3

FIGURE (LEFT). Optogenetic induction of H3.3. A. Primary cultured cerebral cortical neurons were transfected with channelrho-dopsin-2 (ChR2) & stimulated with blue light at 20 Hz (30 sec on, 30 sec off) at 0.25 mW for 2 hr, then analyzed for H3.3A and H3.3B mRNA by qPCR. Control cultures were transfected but not light stimulated (N=3/group). B. HSV-ChR2 or -GFP was injected into mouse mPFC. Two days later, when trans-gene expression is maximal, mPFC was stimulated with blue light as described (17): 10 Hz (3 min, 3 min off) at 2 mW (at tip of fiber-optic) for 15 min daily for 5 days. On Day 6, H3.3 expression was measured by qPCR. Note H3.3A and 3B induction in vivo; H3.3B was also induced in cultured neurons, with a trend for H3.3A induction. Data are mean ± sem (N=6/group). *p<0.05 by ANOVAs and t-tests.

FIGURE (RIGHT). Regulation of H3.3 expression by chronic stress. A. CSDS (as in Fig. 2) induced H3.3 protein levels in mPFC, an effect seen in susceptible (Susc) but not resilient (Res) mice. B. In contrast, CSDS has no effect of H3.3 levels in NAc of normal mice, but induced H3.3 in those subjected to handling in early life (which is protective in the CSDS model), an effect not seen in mice subjected to maternal separation (which promotes susceptibility). Data are mean ± sem (N=8/group); *p<0.05 by ANOVA and t-tests.

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