Functional Analysis of Locus-Specific Pericentric Satellite Expression

PROJECT SUMMARY
The intent of this proposed research is to contribute to the basic understanding of the structure
and function of RNA that is expressed from tandemly repeated regions of the human genome.
Due to recent advances in sequencing of a complete human genome, the tandemly repeated
DNA sequences residing within the centromere and the adjacent regions (the pericentromere)
have only recently been fully characterized. It is known that these abundant tandem repeat
sequences, which constitute 6.2% of the human genome, typically remain silent or are only
expressed at low levels in normal cells. However, in cancer cells, HSATII, a tandemly repeated
pericentric satellite sequence, is aberrantly transcribed into RNA. In these cells, HSATII RNA
accumulates in the nucleus, adjacent to its site of transcription, where it recruits, and potentially
sequesters, nuclear regulatory proteins. While HSATII DNA is found on many different human
chromosomes, only a few of these locations transcribe HSATII RNA, suggesting that the
regulation and sequence composition may vary from one chromosome to another. With the new
resource of a complete sequence map of these pericentric regions, we can now characterize the
chromosomal locations from which HSATII RNA is transcribed in cancer cells for the first time.
In order to study expressed HSATII sequences, long-read sequencing of nuclear expressed
RNA will be mapped to chromosome-specific HSATII variants. It is hypothesized that
expressed HSATII loci will harbor unique sequence motifs. The hypothesis will be tested by
mapping and characterizing expressed chromosome-specific HSATII variants (Aim 1) and by
functional analysis of individual expressed sequence variants in normal cells (Aim 2). The
phenotypic effect of HSATII nuclear accumulation will be accomplished by characterizing the
structure of HSATII RNA and its capacity to bind nuclear regulatory proteins (Aim 3). All of the
proposed research will be conducted by undergraduate students under the close supervision of
the PI, in collaboration with other leaders in genomic and RNA structural analyses. Thus, the
proposed project promises to engage and train undergraduate researchers in innovative
genomics, cytological, and RNA structural probing techniques, which will propel future careers
in genomics and biomedical research.

Grant Number: 2R15GM134495-02A1
NIH Institute/Center: NIH
Principal Investigator: Dawn Carone