Expression and activity of Myc network proteins during cell cycle progression and differentiation

Transcription factors of Myc network are the key regulators of cell proliferation, cell death and differentiation. Identified as a transforming oncogene, myc is the driving force of a variety of human cancers, and inactivation of myc in tumors can cause their rapid regression. Myc network includes the Mad family of proteins, which functionally antagonize Myc and block Myc-induced oncogenic transformation. myc and mad genes are generally expressed in an exclusive fashion proliferating cells express myc, while quiescent and differentiated cells express mad. Myc and Mad antagonistically regulate transcription, at least in part, by recruiting enzymes that respectively acetylate and deacetylate histones at the promoters of their target genes. Histone de/acetylation is thought to affect chromatin structure controlling the access of DNA to the basal transcription machinery.

I: Myc can be aberrantly activated in human cancers by translocations, amplifications or point mutations. N-Myc amplification in rieuroblastomas is a diagnostic marker for tumor progression. During differentiation of neuroblastoma cells N-myc is downregulated, but expression of other Myc network genes has not been studied. We analyzed expression and DNA binding of Mad and Mnt during differentiation of neuroblastoma cells. Our data show that mad genes are induced and downregulated in a transient manner, while mnt is expressed at steady levels. In vitro DNA binding activity of Mnt is also unaffected during differentiation. Ectopic expression of Mad1 does not alter morphological differentiation of neuroblastoma cells. In agreement with previously published data, our results suggest that the activity of Mad proteins might be required only transiently in the differentiation process.

II: Oncogenic transformation by Myc relies on several activities of this protein, including its ability to immortalize cells. Myc has been shown to induce expression of the catalytic subunit of the telomerase enzyme (TERT), believed to be responsible for immortalization of human tumor cells. We demonstrate that Myc interacts in vivo with the hTERT promoter and that Myc binding is associated with histone acetylation. In differentiated cells Mad1 binding to this promoter is associated with histone cleacetylation and downregulation of hTERT expression. We also find that inhibition of histone deacetylase activity derepresses hTERT in normal human cells. Our data implicate Myc and Mad as important regulators of the hTERT expression, and suggest that histone acetylation is instrumental in controlling telomerase activity. Also, our observations provided in vivo evidence for direct binding of Myc and Mad to the same promoter and suggested that their transcriptional targets are overlapping.

III: To further compare DNA recognition by Myc and Mad, we exchanged the Myc basic domain, which defines the DNA-binding specificity, with the corresponding region of Mad I. The resulting chemeric protein was compared with the wildtype c-Myc in oncogenic transformation, regulation of cell proliferation, induction of apoptosis, activation of chromosomal gene expression, and direct binding to chromosomal sites. We find that the wild-type c-Myc and c-Myc(Mad1BR) proteins have identical biological activity and target gene recognition in vivo. Our data are consistent with a model in which Myc and Mad I regulate a common set of target genes.

IV: While tire role of Myc has started to be uncovered, functions of other Myc network proteins remain largely obscure. The Mot protein, in contrast to Mad, is coexpressed with Myc in proliferating cells and is a potential modulator of Mm functions. We characterized some aspects of Min activity in cell cycle progression. We show that Mnt differently binds alternative E-Box elements during cell cycle of immortalized moose fibroblasts. We also find that Mnt is a phosphoprotein, and that phosphorylation of Mnt can be induced by mitogenic stimulation. Interaction with mSin3, a protein that mediates transcriptional repression by Mot, is transiently inhibited upon entry of quiescent fibroblasts into the cell cycle. Mnt directly binds and recruits mSin3 to the promoter of a Myc target gene, cyclinD2, in G0 but not in the G1 phase. We suggest that transcriptional repression by Mot is briefly turned off (possibly by phosphorylation) at entry into the cell cycle to allow induction of Myc target genes.

List of scientific papers

I. Grynfeld Smith A, Popov N, Imreh M, Axelson H, Henriksson M (2003). Expression and DNA-binding activity of MYCN/Max and Mnt/Max during induced differentiation of human neuroblastoma cells. J Cell Biochem. [Accepted]

II. Xu D, Popov N, Hou M, Wang Q, Bjorkholm M, Gruber A, Menkel AR, Henriksson M (2001). Switch from Myc/Max to Mad1/Max binding and decrease in histone acetylation at the telomerase reverse transcriptase promoter during differentiation of HL60 cells. Proc Natl Acad Sci U S A. 98(7): 3826-31.
https://pubmed.ncbi.nlm.nih.gov/11274400

III. Nikiforov MA, Popov N, Kotenko I, Henriksson M, Cole MD (2003). The Mad and Myc basic domains are functionally equivalent. J Biol Chem. 278(13): 11094-9. Epub 2003 Jan 21
https://pubmed.ncbi.nlm.nih.gov/12538578

IV. Popov N, Henriksson M (2004). The Mnt transcription factor is regulated during cell cycle progression and differentiation. [Submitted]