Relationship between DNA Methylation States and Transcription of Individual Isoforms Encoded by the Protocadherin-α Gene Cluster

280

184

Early life experience is associated with long-term effects on behavior and epigenetic programming of the NR3C1 (GLUCOCOR-TICOID RECEPTOR) gene in the hippocampus of both rats and humans. However, it is unlikely that such effects completely capture the evolutionarily conserved epigenetic mechanisms of early adaptation to environment. Here we present DNA methylation profiles spanning 6.5 million base pairs centered at the NR3C1 gene in the hippocampus of humans who experienced abuse as children and nonabused controls. We compare these profiles to corresponding DNA methylation profiles in rats that received differential levels of maternal care. The profiles of both species reveal hundreds of DNA methylation differences associated with early life experience distributed across the entire region in nonrandom patterns. For instance, methylation differences tend to cluster by genomic location, forming clusters covering as many as 1 million bases. Even more surprisingly, these differences seem to specifically target regulatory regions such as gene promoters, particularly those of the protocadherin α, β, and γ gene families. Beyond these high-level similarities, more detailed analyses reveal methylation differences likely stemming from the significant biological and environmental differences between species. These results provide support for an analogous cross-species epigenetic regulatory response at the level of the genomic region to early life experience.conservation | neuronal plasticity V ariation in early life experience is associated with differences in life-long health and behavioral trajectories in animals as well as humans. For example, differences in maternal care in rats during the first week of life are associated with long-term effects on behavior and brain function that persist into adulthood, including alterations in the stress response (1). In humans, similar effects are observed. For instance, childhood maltreatment associates with development of both externalizing and internalizing personality traits and psychopathology in adulthood (2). The association in both rats and humans of stable developmental phenotypes with early life experience suggests that molecular mechanisms may serve as a memory of these early life experiences in both species. In fact, there is evidence that these long-term effects are, at least in part, mediated by epigenetic alterations in the brain. In particular, recent studies have found aberrant DNA methylation in the NR3C1 (GLUCOCORTICOID RECEPTOR) gene promoter of the hippocampi of both rats and humans associated with differential early life experience (3, 4). Exposure of infant rats to stressed caretakers displaying abusive behavior produced persisting changes in methylation of the BDNF gene promoter in the adult prefrontal cortex (5). Early life stress in mice caused sustained DNA hypomethylation of an important regulatory region of the AVP gene (6).Although explanations involving a single site are appealing, it is unlikely that the broad systemic response to early life experien...

Section: Discussionsupporting

confidence: 81%

“…These methylation differences observed in human hippocampus are of interest because the protocadherin families of genes are known to be regulated by promoter methylation (16)(17)(18) and have been implicated in synaptic function and neuronal connectivity (19)(20)(21)(22).…”

Section: Conserved Methylation Sensitivity In the Protocadherin Familmentioning

confidence: 99%

Conserved epigenetic sensitivity to early life experience in the rat and human hippocampus

Suderman

McGowan

Sasaki

et al. 2012

280

184

“…CTCF binding can be blocked by CpG methylation (24). Pcdh promoter CpG methylation is inversely correlated with Pcdh expression in cell lines and in vivo (6,23,25) and inhibition of CpG methylation by 5-AZT can activate silent Pcdhα isoforms (25). These findings suggest that CpG methylation could regulate CTCF binding to silent promoters, either directly or through competition with other proteins, such as MeCP2, that bind methylated CpGs.…”

Section: Discussionmentioning

confidence: 88%

Role of CCCTC binding factor (CTCF) and cohesin in the generation of single-cell diversity of Protocadherin-α gene expression

Monahan

Rudnick

Kehayova

et al. 2012

139

125

Extraordinary single-cell diversity is generated in the vertebrate nervous system by the combinatorial expression of the clustered protocadherin genes (Pcdhα, -β, and -γ). This diversity is generated by a combination of stochastic promoter choice and alternative premRNA splicing. Here we show that both the insulator-binding protein CTCF and the cohesin complex subunit Rad21 bind to two highly conserved DNA sequences, the first within and the second downstream of transcriptionally active Pcdhα promoters. Both CTCF and Rad21 bind to these sites in vitro and in vivo, this binding directly correlates with alternative isoform expression, and knocking down CTCF expression reduces alternative isoform expression. Remarkably, a similarly spaced pair of CTCF/Rad21 binding sites was identified within a distant enhancer element (HS5-1), which is required for normal levels of alternative isoform expression. We also identify an additional, unique regulatory role for cohesin, as Rad21 binds to another enhancer (HS7) independently of CTCF, and knockdown of Rad21 reduces expression of the constitutive, biallelically expressed Pcdhα isoforms αc1 and αc2. We propose that CTCF and the cohesin complex initiate and maintain Pcdhα promoter choice by mediating interactions between Pcdhα promoters and enhancers.

“…In summary, we show that CTCF binds directly to a repertoire of regulatory sequences within the Pcdh locus in vitro and in vivo and that this binding correlates with Pcdh gene expression. (3,17,18). The core sequences within CSE contain a CpG dinucleotide (2).…”

Section: Resultsmentioning

confidence: 99%

CTCF/cohesin-mediated DNA looping is required for protocadherin α promoter choice

Guo

Monahan

et al. 2012

227

321

The closely linked human protocadherin (Pcdh) α, β, and γ gene clusters encode 53 distinct protein isoforms, which are expressed in a combinatorial manner to generate enormous diversity on the surface of individual neurons. This diversity is a consequence of stochastic promoter choice and alternative pre-mRNA processing. Here, we show that Pcdhα promoter choice is achieved by DNA looping between two downstream transcriptional enhancers and individual promoters driving the expression of alternate Pcdhα isoforms. In addition, we show that this DNA looping requires specific binding of the CTCF/cohesin complex to two symmetrically aligned binding sites in both the transcriptionally active promoters and in one of the enhancers. These findings have important implications regarding enhancer/promoter interactions in the generation of complex Pcdh cell surface codes for the establishment of neuronal identity and self-avoidance in individual neurons.T he clustered protocadherin (Pcdh) genes are expressed in the nervous system and organized into three closely linked clusters (α, β, and γ) (1-5). The human Pcdhα gene cluster contains 13 highly similar variable first exons (α1 to α13) arrayed in tandem and two more distantly related c-type variable first exons designated αc1 and αc2 (Fig. 1A). The variable first exons encode the extracellular, transmembrane, and juxtamembrane intracellular domains of the Pcdhα proteins. Each of these 15 variable first exons is cis-spliced to a single set of three downstream constant exons that encode a distal intracellular domain (1-3). The human β cluster is located downstream from the α cluster and contains a tandem array of 16 highly similar variable exons but with no constant exons, whereas the γ cluster contains 22 variable first exons arrayed in tandem and divided into three types (γa1 to γa12, γb1 to γb7, and γc3 to γc5) (Fig. 1D). As in the case of the α cluster, each of these 22 γ variable first exons is cis-spliced to a single set of three downstream constant exons, which are distinct from the α constant exons, to generate diverse γ mRNAs (1, 3, 6). Analyses of the α and γ transcripts have revealed that highly similar Pcdh alternate isoforms are expressed in a stochastic fashion, whereas all of the c-type divergent isoforms, αc1 and αc2 in the α cluster and γc3, γc4, and γc5 in the γ cluster, are expressed ubiquitously in all cells (1-3, 5, 7). Hereafter, we refer to the c-type genes as "ubiquitously expressed" in contrast to the "alternately expressed" Pcdh genes ( Fig. 1 A and D). A combination of stochastic activation of alternate promoters and constitutive activation of c-type ubiquitous promoters generates enormous single-cell diversity on the surface of individual neurons.Significant advances have been made in understanding the mechanisms by which individual neurons express distinct combinations of the clustered Pcdh genes (2, 3, 8-10). Two long-range cis-regulatory elements in the α cluster, HS5-1 and HS7 (hypersensitive sites 5-1 and 7), function as developmental and tissue...