Volvox carteri: molecular evolution of RegA and related proteins, and Chromatin immunoprecipitation (ChIP) application to investigate RegA targets and modulation of hsp70A promoter chromatin
Creators: name: Howard, Alicia Deanne
Publisher: name: Maryland Shared Open Access Repository
Year: 2013
Subjects: ChIP-Seq
Chromatin immunoprecipitation
hsp70A
RegA
VARL genes
Volvox carteri

description: Research seeking to understand the molecular innovations involved in transitioning from unicellular to multicellular life has been limited by a lack of closely related unicellular and multicellular organisms able to serve as tractable model systems. The volvocine green algae overcome such limitations.  Its members include &lt;italic&gt;Chlamydomonas reinhardtii&lt;/italic&gt; (unicellular) and &lt;italic&gt;Volvox carteri&lt;/italic&gt; (multicellular), which shared a common ancestor ~200 million years ago. &lt;italic&gt;V. carteri&lt;/italic&gt; contains two cell types: reproductive gonidia and motile, terminally differentiated somatic cells.  The &lt;italic&gt;regA&lt;/italic&gt; gene product (RegA) is required for somatic cell fate and, when mutated, produces the Reg mutant phenotype.  Reg mutant somatic cells develop normally during the first day of the life cycle, but dedifferentiate, then develop into gonidia.  This conversion from spatial to temporal cell differentiation makes RegA an intriguing candidate for elucidating the molecular origins of one key aspect of multicellularity in the volvocine algae. Using comparative genomics, the products of &lt;italic&gt;regA&lt;/italic&gt; and &lt;italic&gt;regA&lt;/italic&gt;-like (&lt;italic&gt;rls&lt;/italic&gt;) genes were found to contain a putative DNA-binding domain (VARL, for Volvocine-Algal-RegA-Like), also encoded by several &lt;italic&gt;C. reinhardtii&lt;/italic&gt; genes.  This dissertation addresses the evolutionary origins of RegA structure and function, as well as its role as a putative transcription factor.  Using Rt-PCR I discovered that the &lt;italic&gt;rlsA&lt;/italic&gt;, &lt;italic&gt;rlsE&lt;/italic&gt;, &lt;italic&gt;rlsF&lt;/italic&gt;, and &lt;italic&gt;rlsH&lt;/italic&gt; mRNAs are spliced to produce VARL-domain proteins; however, &lt;italic&gt;rlsC&lt;/italic&gt; and &lt;italic&gt;rlsG&lt;/italic&gt; mRNAs are not.  To address a possible role for RegA as a transcription factor, I generated a plasmid encoding a GFP-RegA fusion, which rescued &lt;italic&gt;regA&lt;/italic&gt; loss of function mutants.  I then optimized a Chromatin Immunoprecipitation (ChIP) platform for use in &lt;italic&gt;V. carteri&lt;/italic&gt;, and validated ChIP by examining histone occupancy at the &lt;italic&gt;hsp70A&lt;/italic&gt; promoter.  I discovered that heat shock decreases unmodified histone H3 occupancy; increases relative H3k4me1, H3K14ac and pan-acetylated histone H4 levels; but has no effect on the relative H3K4me3 levels.  I used ChIP, followed by massively parallel sequencing (ChIP-Seq), to identify DNA targets of RegA.  No statistically significant sequences were identified, but non-statistically significant enrichment was detected at the promoter and eleventh intron of the &lt;italic&gt;retinoblastoma-related 1&lt;/italic&gt; (&lt;italic&gt;rbr1&lt;/italic&gt;) gene.  I confirmed enrichment by ChIP-qPCR, suggesting that RegA might regulate rbr1 during somatic cell terminal differentiation.