Reversible post-transcriptional modifications (PTMs) to core histone proteins (H2a, H2b, H3 and H4), such as methylation, acetylation and phosphorylation, aid in determining the transcriptional status of the genes at which they occur. For example, high levels of acetylation are associated with gene activation whereas low levels lead to silencing. Gene expression is also regulated by both the positional and combinatorial status of PTMs.
Transcription factors (TFs) have also been demonstrated to undergo many of the same PTMs as histones [1] but the functional consequences of these are much less well understood. We are therefore seeking to investigate, whether there are temporal changes in the type and location of PTMs on a series of key transcription factors during a developmental trajectory. To this end we have chosen the G1E-ER4 cell line [2], an erythroid precursor line containing a tamoxifen inducible copy of the master TF GATA1. This environment has been selected because generally, the differentiation of stem cells into distinct lineages leads to significant changes in histone marks and we hypothesize that this will be the case for TFs [3].
To allow isolation of the TFs of interest, we have employed CRISPR/Cas9 to introduce a FLAG tag and flexible Gly-Ser-rich linker to the c-terminus of each TF. TAL1 and KLF1 have been successfully tagged. FLI1, BCL11a, PU1 have also undergone CRISPR-ing to introduce the tag and linker. The success of this engineering is currently being assessed.
TAL1 has been successfully immunoprecipitated from the nuclear fraction of differentiated and undifferentiated cells and the resulting protein subjected to LC-MS/MS to determine the presence and location of PTMs. In the first instance we are looking for acetylation and phosphorylation.
Overall, we aim to establish the importance of TF PTMs in the expression of genes and to determine whether a combined histone/TF code exists.