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Y.-H. Liu, F. F. Zhuang, S. C. Yiu, R. E. Smith; Mll, a Mammalian Homolog of the Drosophila trithorax Gene, Functions as a Transcriptional Repressor on the Msx2 Promoter. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3210.
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Msx2 is an important transcriptional regulator for eye development. In the developing eye, Msx2 expression is spatially and temporally controlled and it helps fine-tuning the expression level of Bmp4. Msx2 overexpression or inactivation has a direct effect on Bmp4 transcription in the developing eye and leads to microphthalmia and dysmorphogenesis of the lens and retina. Therefore, a precise mechanism that operates at the transcriptional level must be in place to regulate Msx2 expression. Several transcriptional activators of Msx2 have previously been identified that include BMP-restricted Smads and Wnt signaling mediators, TCFs. Factors that repress its expression remain elusive.
In previous microarray studies, Mll (Mixed-lineage leukemia) was identified as a potential repressor of Msx2 transcription. Mll, a mammalian homolog of Drosophila trithorax (trx) gene, encodes a histone H3 Lys-4 specific methyltransferase. To demonstrate that Mll functions as a direct regulator of Msx2 transcription, we utilized Msx2-promoter lacZ reporter s and cell lines established from Mll wildtype (Mll+/+ MEF) and null embryonic fibroblasts (Mll-/- MEF) to perform transfection studies. These Msx2-promoter reporter plasmids were constructed utilizing either its own promoter or a heterologous promoter from Hsp70.
Results from our transfection studies showed unequivocally that Mll is a powerful repressor that acts on the Msx2 promoter. To identify the sequences that respond to repression by Mll, we transfected various Msx2-lacZ fusion constructs into Mll+/+ MEFs and Mll-/- MEFs and compared the transcriptional responses of these constructs in the presence or absence of Mll. Every construct that contains a 512bp fragment proximal to the transcriptional initiation site showed nominal transcriptional activity in Mll+/+ MEFs but exhibited high transcriptional activity in Mll-/- MEFs. A construct that lacks the proximal promoter showed equivalent transcriptional activity in either the wiltype Mll cell line or the Mll null cell line. Further deletion of this 521bp fragment to within 429bp did not improve transcriptional response to Mll.
Mll functions as a potent transcriptional repressor on the Msx2 promoter in a sequence dependent manner. The implication of this novel finding is that silencing of Msx2 transcription during embryonic development may require modification at the chromatin level.
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