Figure 8. The YY1-dependent repression of Tcf4 and Id4 is lineage-specific and mediated by recruitment of HDAC1 to their promoters.

(A) Quantitative RT-PCR of Tcf4, Id4 and REST in oligodendrocyte progenitors transfected with pCX-EGFP (vector) or pCX-yy1-EGFP (YY1) and induced to differentiate into astrocytes (astro) or oligodendrocytes (oligo). The transcript levels of each gene in vector transfected cells was arbitrarily set as 100; *p<0.05. (B) Co-immunoprecipitation of YY1 and HDAC-1 and -2. Western blot analysis of whole cell lysates (lysates) and YY1 immunoprecipitated protein extracts (IP:YY1) derived from undifferentiated progenitors (prog) and cells differentiated into oligodendrocytes (diff prog) or astrocytes (astro). (C) YY1 activity, measured by TransLucent vector reporter system. Note the increased activity of YY1 in cultures of progenitors differentiating into oligodendrocytes (diff. prog.) compared to undifferentiated cells (prog.) or cells differentiating into astrocytes (astro). **p<0.01. (D) Chromatin immunoprecipitation (ChIP) of samples isolated from progenitors (prog), differentiating oligodendrocytes (diff) or astrocytes (astro) and immunoprecipitated with antibodies against YY1 and HDAC1. The diagram shows the Tcf4 promoter with the relative position of the YY1 consensus sequences (black boxes) and the regions of DNA (roman numerals) amplified by specific primer pairs (arrows). Input DNA was used as positive control, while ChIP in the absence of antibodies or amplification of immunoprecipitated chromatin with primers for region IV were used as negative controls. (E) ChIP of samples isolated in the same conditions described above. The diagram shows the Id4 promoter, with the position of the YY1 consensus sequence (black box) and the regions of the promoter (roman numerals) amplified by specific primer pairs (arrows). YY1 was bound to the Id4 promoter in progenitors, but it recruited HDAC1 only when cells differentiated into oligodendrocytes. No binding was observed in progenitors differentiating into astrocytes. (F) Model of oligodendrocyte progenitor differentiation as two step event. First, proliferating progenitors exit from the cell cycle and remain in an undifferentiated state characterized by high levels of transcriptional inhibitors (Id4, Tcf4) and lack of myelin gene expression. As the progenitors begin differentiating, repressive complexes containing YY1 and HDAC1 are recruited to the promoter of these inhibitors. The decreased levels of these inhibitory molecules allow myelin gene expression to begin.