CTCF Tucks Genes Into Their Lamina Associated Beds
The circadian rhythm allows cells to synchronize across a 24-hour clock. When the clock is disrupted individuals become predisposed to complex diseases like cancer, psychiatric disorders, diabetes, and metabolic syndrome. During the ticking of the clock oscillations in the expression of a number of genes, for instance those involved in metabolism, are controlled by chromatin remodelling. Chromatin architecture can be quite complex and involves forming regulatory structures in large scale domains. One example of this type structure is lamina associated domains (LADs). These LADs reside at the periphery of the nucleus and often contain genes that are developmentally silenced. LADs are also associated with repressive H3K9me2 and H3K9me3 domains that are referred to as large organized chromatin K9-modifications (LOCKs). However, the mechanistic relationship between clock and LOCK has remained a mystery.
Repressive LOCKs are only one side of the regulatory coin as there are also activating domains that are created by regulatory proteins. CTCF is a genomic insulator that creates distinct chromatin domains by bringing distant genomic sequences together. Interestingly, CTCF’s function as an insulator is dependent on PARylation by PARP1, a gene known to be involved in the circadian rhythm!
Using the circularized chromosome conformation capture (4C) technique, a team from the Karolinska Institutet in Sweden investigated the chromatin interactions in human embryonic stem cells (hESCs) and derived embryoid bodies (hEBs). This allowed them to characterize the chromatin interactions associated with clock-controlled genes and also the key proteins involved. They chose to use the H19 imprinting control region (ICR) as bait since it is in a domain controlled by the clock. They found 518 regions that interact with the H19 ICR, with a majority of these interactions being inter-chromosomal and developmentally regulated. These interactions were confirmed by cross-referencing the the physical distances between sequences with 3D DNA fluorescence in situ hybridization (FISH). The sequences identified by 4C included genes as well as intergenic regions and were enriched for functions related to cell adhesion and synaptic processes. Interestingly, active circadian genes were coming into contact with LADs within the same interacting chromatin modules. The team also utilized a PARP inhibitor (olaparib) and used a number of other techniques to show that PARP1 and CTCF are critical in maintaining the chromatin contacts between domains at the H19 ICR, implicating them in circadian rhythm regulation.
Next, with the knowledge of the players involved in the strange inter-chromosomal contacts, the team turned to HCT116 (colon cancer) cells since the hESCs can present challenges when trying to control the clock. Taking advantage of the different cell line, they used serum shock to synchronize and reboot the circadian oscillations in order to see what happens to chromatin structure when the circadian clock starts ticking. The team visualized the locations of CTCF-PARP1 using an in situ proximity ligation assay (ISPLA) and found that genomic interactions oscillated across a 24-hour phase! Additionally, the CTCF-PARP1 interaction was preferentially located at the lamina of the nucleus, a pattern not seen in CTCF-CTCF or PARP1-PARP1 interactions. Using 3D DNA FISH, the team found that H19 co-localized with the nuclear lamina in a rhythmic pattern, with discrete peaks. Next the team examined what would happen when these two key genes were interfered with. They used olaparib for PARP1 and siRNAs for CTCF or PARP1, and found that disruption of CTCF and PARP1 prevented the circadian recruitment of a key gene (PARD3) to the nuclear lamina. This experimentation helped reveal that both CTCF and PARP1 are critical to the dynamic chromatin positioning behind the circadian rhythm.
Finally, the team examined how circadian oscillations in chromatin structure regulate transcription. The team noticed that when PARD3 was recruited to the lamina it still had high transcriptional activity at first and that the repression of transcription took some time, suggesting that recruitment to the lamina precedes transcriptional repression. In order to figure out what was causing the repression, the team investigated H3K9me2 domains, also known as LOCKs, as they are enriched for in the lamina. Using single-cell analysis they found that H3K9me2 was highest at the time of transcriptional attenuation, which was some time after the recruitment to the nuclear lamina. Later on, low levels of the mark coincided with higher transcription and a different nuclear position. Solidifying the role of LOCKs in silencing constitutive LADs, the team used a small molecule inhibitor of the histone methyltransferase G9a/Glp to prevent the deposition of inhibiting H3K9me2, which removed the circadian oscillations in transcription. These findings show that there is a bit of lag between movement to the lamina and the addition of large-scale repressive marks that silence transcription.
Taken together these experiments suggest a mechanism where active sequence is recruited to the lamina by PARP1 and CTCF, and after a bit of time repressed LOCKs are formed by G9a/Glp. Once the interaction between CTCF and PARP1 degrades, the sequence is released back into the nucleus where the repressive marks eventually degrade in a timely manner. Ultimately, this research reveals an inter-chromosomal network connecting active circadian genes to repressive LADs and LOCKS, which provides novel insight into the mechanisms behind the repressive arm of the circadian clock. It also implicates CTCF in the oscillations of the circadian rhythm and shows that dynamic chromosomal positioning keeps the circadian clock ticking.
Zhao H, Sifakis EG, Sumida N, Millán-Ariño L, Scholz BA, Svensson JP, Chen X, Ronnegren AL, Mallet de Lima CD, Varnoosfaderani FS, Shi C, Loseva O, Yammine S, Israelsson M, Rathje LS, Németi B, Fredlund E, Helleday T, Imreh MP, & Göndör A (2015). PARP1- and CTCF-Mediated Interactions between Active and Repressed Chromatin at the Lamina Promote Oscillating Transcription. Molecular cell, 59 (6), 984-97 PMID: 26321255
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