The following Figure shows sequence conservation between human and mouse in the
ID: 69079 • Letter: T
Question
The following Figure shows sequence conservation between human and mouse in the top panel, and ChIP-seq results from developing mouse hindlimb for occupancy of Pitx1 protein on DNA in panel B.
Of the three Regions (1, 2, and 3), which one(s) is (or are) most likely acting as (an) enhancer(s) in the tissue that was harvested for the ChIP-seq experiment? How do you know this? (2 pt)
How could you test your hypothesis from part A using an in vivo experiment? (1 pt)
Why might Region 3 show such high conservation between human and mouse, yet show very little Pitx1 ChIP-seq signal? (1 pt)
Explanation / Answer
Mammalian development requires the precise spatial and temporal control of gene expression. Much of this regulatory information is encoded in thousands of cis-acting elements that are distributed across the genome, often at great distances from their target genes . The mechanisms that connect cis-regulatory elements to their specific targets and that prevent them from inappropriately influencing other genes are not well defined. Recent studies suggest cohesin stabilizes DNA loops between distant-acting enhancers and their target promoters. Cohesin is a ring-shaped complex consisting of the core subunits SMC1A, SMC3, SCC1. Although cohesin does not bind DNA directly, it colocalizes with tissue-specific transcription factors on chromatin and is thought to stabilize binding of transcription factors at enhancers. In embryonic stem cells, cohesin shows cell-type-specific binding at enhancers and promoters that engage in cell-type-specific interactions, and knockdown of cohesin results in aberrant gene expression and loss of pluripotency. These studies suggest tissue-specific gene activation is the result of tissue-specific cohesin-mediated DNA looping events.
Additionally, cohesin has been shown to associate with the insulator factor CTCF, which targets cohesin to specific sites in the genome. Cohesin is required for the enhancer-blocking functions of CTCF binding. CTCF also establishes chromatin barriers to prevent the spread of heterochromatin. In embryonic stem (ES) cells, chromatin loops mediated by CTCF interactions show correlated patterns of active or repressed histone modifications contained or excluded by the loop CTCF shows largely invariant binding patterns across tissues and, in conjunction with cohesin, may establish constitutive chromatin topologies in the nucleus.
Despite these findings, global insight into the role of cohesin in gene regulation remains limited because cohesin-mediated interactions have yet to be mapped at a genome-wide scale. Here, we use chromatin interaction analysis with paired-end tag sequencing (ChIA-PET) to detect putative regulatory interactions involving the cohesin subunit SMC1A in the embryonic mouse limb. The limb is particularly well suited for this purpose. Distant-acting enhancers are essential for limb development. Moreover, a large number of distant-acting enhancers in the limb have been experimentally characterized by chromatin mapping and mouse transgenic assays, providing a functional basis for interpreting cohesin-mediated interactions . Previous ChIA-PET studies have been performed in mouse and human cell culture to capture interactions involving transcription factor estrogen receptor-, CTCF, and RNAPII . However, cohesin is recruited to both insulators and enhancers, suggesting it is involved in diverse regulatory interactions. Our ChIA-PET analysis of cohesin-associated interactions in developing limb revealed tissue-specific enhancer-promoter interactions, as well as interactions involving both cohesin and CTCF that potentially establish constitutive chromatin domains across tissues. Surprisingly, we also identified interactions that are maintained in multiple tissues between promoters and distal regulatory elements that show tissue-specific activation or repression during development.