Abstract

CCCTC binding factor (CTCF) is involved in organizing chromosomes into mega base-sized, topologically associated domains (TADs) along with other factors that define sub-TAD organization. CTCF-Cohesin interactions have been shown to be critical for transcription insulation activity as it stabilizes long-range interactions to promote proper gene expression. Previous studies suggest that heterochromatin boundary activity of CTCF may be independent of Cohesin, and there may be additional mechanisms for defining topological domains. Here, we show that a boundary site we previously identified known as CTCF binding site 5 (CBS5) from the homeotic gene cluster A (HOXA) locus exhibits robust promoter activity. This promoter activity from the CBS5 boundary element generates a long noncoding RNA that we designate as boundary associated long noncoding RNA-1 (blncRNA1). Functional characterization of this RNA suggests that the transcript stabilizes long-range interactions at the HOXA locus and promotes proper expression of HOXA genes. Additionally, our functional analysis also shows that this RNA is not needed in the stabilization of CTCF-Cohesin interactions however CTCF-Cohesin interactions are critical in the transcription of blncRNA1. Thus, the CTCF-associated boundary element, CBS5, employs both Cohesin and noncoding RNA to establish and maintain topologically associated domains at the HOXA locus.

Highlights

  • CCCTC binding factor (CTCF) is an eleven zinc finger protein that affects gene expression by a variety of mechanisms [1,2,3]

  • Our initial characterization of boundary activity at the HOXA locus on chromosome 7 started with reporter assays to determine if the HOXA boundary element, CTCF binding site 5 (CBS5), exhibited any other activities besides the boundary activity that we reported previously

  • When CBS5 is cloned in the same orientation as the other HOXA genes (HOXA7 and HOXA9), CBS5 (-), the reporter exhibited 70% of the promoter activity observed from the SV40 Promoter/Enhancer control reporter (Fig 1B)

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Summary

Introduction

CCCTC binding factor (CTCF) is an eleven zinc finger protein that affects gene expression by a variety of mechanisms [1,2,3]. CTCF was characterized as a transcription repressor of the c-Myc promoter [4]. Later studies have shown that CTCF can act as a transcription activator [5]. CTCF regulates transcription of the XIST gene and RNA interactions made with CTCF are critical for X chromosome inactivation (XCI) [6,7,8]. Cohesin has been found to bind to a majority of CTCF binding sites (CBSs) across the genome [9]. CTCF-Cohesin interactions are PLOS ONE | DOI:10.1371/journal.pone.0136104. CTCF-Cohesin interactions are PLOS ONE | DOI:10.1371/journal.pone.0136104 August 24, 2015

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