3D Chromatin Organization, Transcription, and Behavior
The chromosomes of differentiated cells contain heterochromatic and euchromatic compartments. Heterochromatin is highly condensed and associated with repressed transcription, whereas in euchromatin transcription is generally active. Spatial organization of heterochromatin can vary vastly among different differentiated cell types1. In contrast to differentiated cells, embryonic stem cells contain only sparse heterochromatic regions. These observations support the idea that structural changes in 3D chromatin organization participate in the epigenetic regulation of gene expression, though the mechanisms are poorly understood. In a recent study, Ito et. al. generated a bitransgenetic mouse expressing a chimeric, GFP-tagged histone H2B – specifically expressed in CaMKIIa+ (calmodulin-dependent protein kinase II A) principal neurons in the forebrain. The encorporation of this chimeric protein into the DNA of mature neurons was able to disrupt heterochromatin organization. Additioanlly, they also show that this change in chromosomal architecture is coupled with dysregulation of serotonin receptor genes – as well as the particular behaviors modulated by the serotonin system2.
Telomeres, centromeres, and their adjacent chromatin regions are highly heterochromatic and marked with CpG and histone methylation. Centromeres, and therefore centromeric and pericentromeric chromatins, often cluster together into “chromocenters” in the nucleus. “Peripheral” heterochromatin describes heterochromatin attached to the inner nuclear membrane. The Ito group found that the 3D organization of the different heterochromatin regions described above were disrupted in neurons containing the chimeric H2BGFP protein. When stained with DAPI, a molecule that is able to bind to and fluorescently label DNA, H2BGFP neurons showed a reduction of chromocenters and an increase in irregular DNA foci. Consistently, fluorescence in situ hybridization (FISH) using a pancentromeric probe revealed abberant 3D organization – instead of overlapping with chromocenters, the probes in H2BGFP neurons yielded small dots scattered within the DNA foci. Staining for histone and cyosine methylation, modifications associated with heterochromatin, also showed disruption in patterning and was accompanied by an overall decrease in the DNA methylation. Additionally, peripheral heterochromatin was seen detached from the nuclear membrane, causing nuclear laminar invagination. These results are supportive of H2BGFP’s role in declustering pericentromeric chromatin, causing abnormal heterochromain-nuclear laminar interactions, and reorganizing transcriptionally active domains – likely due to it’s bulky GFP tag.
Subsequent Microarray analysis of global gene expression showed surprisingly modest changes in H2BGFP neurons. Perhaps contrary to what might have been expected, disorganization of heterochromatin did not lead to activation of normally repressed genes. In fact, most of the changes detected were reductions in expression levels. Interestingly, a significant amount of the differentially down-regulated genes in H2BGFP neurons, were genes responsible for neuronal functional and behavior, including three serontonin receptor genes Htr1a, Htr1b, Htr2a, as well as Drd5 – a dopamine receptor gene, and Npy2r – a neuropeptide receptor gene. The athuors used the analysis proram, Gene Ontology, to examine the possible functional implications of the changes in expression profiles. Gene Ontology predicted impaired glutamatergic synaptic transmission, serotonin receptor signaling and dopamine secretion. Abnormal anxiety related response, locomotor activation, spatial learning and hyperactivity were also predicted.
Behavioral tests were conducted to validate the predictions made based on the expression profiles. Locomotor hyperactivity was observed in H2BGFP mice as measured by an increase in ambulatory distance, faster speed, and reduced resting time. The transgenic mice also showed reduced responsiveness to stimuli, manifested as decreased marble burying behavior, increased latency in novel object exploration and hot plate task, impaired social interaction and sensorimotor gating. They also performed significantly worse in the hidden water maze task, which assesses spatial memory and learning. Overall, the behavioral observations are consistent with the neuronal gene expression profile!
Of the genes identified, the most affected and clinically relevant was the gene encoding the serotonin 1A receptor subtype (Htr1a). In situ hybridization showed altered spatial distribution of Htr1a loci in hippocampal CA1 neurons. In control animals, the two loci are located at the nuclear margin and interior. In H2BGFP mice, due to the disrupted perinuclear and pericentrometric heterochromain structure, the Htr1a loci were found located near or within aberrant DNA foci. Colocalization of Htr1a and activated RNA polymerase II was also reduced in transgenic animals. Using electrophysiological recordings, it was also determined that serotonin signalling was reduced in the H2BGFAP-expression CA1 neurons. These results indicated the involvement of impaired serotonin signaling system in the behavioral deficits observed in H2BGFP transgenic animals.
It has been previously reported that the chimeric H2BGFP does not interfere with the chromatin structure of dividing cells – however, Ito et al. describes a mechanism by which not only does H2BGFP reorganize the 3D structure of chromatin, but that the changes lead to altered gene expression profiles and quantifiable behavior outputs. The authors also noted that this chimeric protein affects different neuronal types to various degrees. How 3D chromatin structure plays into the larger picture of cell fate determination and normal cellular function is largely unknown. New methods are needed to provide high resolution 3D chromatin structure visualization which will allow for greater ability to describe as well as predict quantitative relationships between 3D structural changes and genetic expression profiles – the developing of these technologies ultimately in efforts to one day be able to manipulate the 3D chromatin structure for favorable outcomes.
1 Politz JC, Scalzo D, & Groudine M (2013). Something silent this way forms: the functional organization of the repressive nuclear compartment. Annual review of cell and developmental biology, 29, 241-70 PMID: 23834025
2 Ito S, Magalska A, Alcaraz-Iborra M, Lopez-Atalaya JP, Rovira V, Contreras-Moreira B, Lipinski M, Olivares R, Martinez-Hernandez J, Ruszczycki B, Lujan R, Geijo-Barrientos E, Wilczynski GM, & Barco A (2014). Loss of neuronal 3D chromatin organization causes transcriptional and behavioural deficits related to serotonergic dysfunction. Nature communications, 5 PMID: 25034090