CGGBP1 from higher amniotes restricts cytosine methylation and drives a GC-bias in transcription factor binding sites at repressed promoters [Array]

Human and other vertebrate representative forms of CGGBP1 have been used in this study to assay global gene expression. HEK293T cells with endogenous CGGBP1 knocked down were used to over-express other vertebrate representative forms of CGGBP1 (Latimeria chalumnae (Lc), Anolis carolinensis (Ac), Gallus gallus (Gg)) followed by RNA extraction and one-coloured global gene expression analysis (Agilent). HEK293T cells with normal level of endogenous CGGBP1 were used to over-express Human CGGBP1 (Hs) or pcDNA3.1(+) empty vector (Ev). Overexpression of CGGBP1 forms resulted in the repression of specific gene sets at two different temperatures (37 degrees and 40 degrees Celsius). The proximal promoters of these repressed genes exhibit a marked difference in the occurrence of GC-rich transcription factor binding sites (TFBSs). Through additional studies on these samples we have separately assayed global cytosine methylation patterns (submitted separately to NCBI GEO) and reported that the GC content of these promoters is determined by a combination of motif occurrence as well as motif GC contents. Notably, the within these gene promoters human CGGBP1 was found to restrict cytosine methylation at certain GC-rich TFBSs. A high-throughput analysis of the GC compositional bias across these CGGBP1-regulated TFBSs, using orthologous sequences from over 100 species, revealed a strong link between CGGBP1-induced cytosine methylation restriction and GC retention at specific TFBSs. Further, orthology analysis showed that this regulatory mechanism of CGGBP1 is conserved in higher amniotes (birds and mammals) and exhibits lineage-specific variations in lower amniotes (reptiles). To investigate gene expression, HEK293T cells were transfected with various CGGBP1 forms, including those from Latimeria chalumnae (Lc), Anolis carolinensis (Ac), Gallus gallus (Gg), and Homo sapiens (Hs), as well as with the pcDNA3.1(+) empty vector control. Gene expression was assessed 72 hours post-transfection. In parallel, heat stress conditions were applied by first incubating the cells at 37°C for 24 hours, followed by 48 hours of continuous heat stress at 40°C. Gene expression changes were subsequently analysed to evaluate the impact of heat stress. All experiments were performed in triplicate to ensure statistical reliability.