Regulation: A conserved DNA element with the ability to regulate the entire INK4/ARF locus has been identified in close proximity of the locus and named regulatory domain (RD). It appears to promote transcriptional repression of all three genes encoded by the locus, in a manner dependent on CDC6. In proliferating embryonic fibroblasts (MEFs), EZH2 a member of the polycomb repressive complex 2 (PRC2) as well as BMI1 and M33 members of the polycomb repressive complex 1 (PRC1) are strongly expressed and are found to localize to the INK4/ARF RD. BMI1 has been shown to interact specifically with CDC6. These polycomb group (PcG) complexes repress the locus activity through the establishment of repressive chromatin modifications such as H3K27 trimethylation. During senescence, binding of these complexes to RD is lost and correlates with increased expression of the INK4/ARF genes (Figure 1).
The p15INK4B gene is also silenced by a long non coding RNA, called antisense non-coding RNA in the INK4 locus (ANRIL), whose expression was found to be inversed to the expression of p15INK4B in leukemia cell lines. It was shown that ANRIL induces the silencing of p15INK4B in cis and trans by triggering heterochromatin formation in a Dicer-independent manner. PcG complexes are recruited to the INK4/ARF locus by ANRIL and modulate its repression (Figure 1). Additionally, a naturally occurring antisense circular ANRIL RNAs (cANRIL) has also been described. Different forms of cANRIL are produced in most INK4/ARF expressing cells, suggesting that alternative splicing events leading to different ANRIL structures can contribute to changes in PcG-mediated INK4/ARF repression.
Specific transcription regulators of p15INK4B have also been reported (see Figure 2). These include TGF-b, MIZ-1, SMAD3/SMAD4 complex, SP1, c-MYC, IRF8, PU.1, SNAIL and EGR1 factors among others.
p15INK4B expression is dramatically induced by TGF-b, suggesting that it is a potent downstream effector of TGF-b mediated growth arrest.
MIZ-1 is a transcription factor that has been shown to bind the initiator element (Inr) in the promoter region and induce the transcription of p15INK4B in epithelial cells. However it can also recruit transcriptional co-repressors such as c-MYC and GFI-1 to the promoter region by binding to them and forming inhibitory complexes. TGF-b has been reported to re-activate the core promoter through downregulation of C-MYC and GFI-1, thereby releasing endogenous MIZ-1 from inhibition.
The SMAD3/4 complex readily forms following TGF-b treatment and physical interaction between this complex, MIZ-1 and promoter-bound SP1 protein has been described. These interactions have been proposed to constitute a platform for the recruitment of co-activators, and do not seem to be affected by the suppressor activity of c-MYC. The inhibitory function of c-MYC seems to be cell-type dependent, as it was confirmed in epithelial cells but not in the hematopoietic lineage. In myeloid cells, the transcription factor c-MYB was shown to prevent the transcription and the upregulation of p15INK4B which is normally associated with the differentiation process. The mechanism by which C-MYB does this is unclear but it is not through upregulation of c-MYC, a known target of c-MYB.
A tri-component transcriptional complex consisting of SNAIL, SP1 and EGR-1 was also described for its ability to trigger the p15INK4b promoter activation upon TPA treatment.
In murine myeloid cells specifically, the interferon consensus sequence-binding protein/interferon regulatory factor 8 (ICSBP/IRF-8) in combination with PU.1 were shown to bind p15Ink4b promoter and activate the transcription of the gene in response to IFN-b treatment.
In AML patients with inv(16), p15INK4B silencing was found to be caused by inv(16)-encoded core binding factor beta-smooth muscle myosin heavy chain (CBFb-SMMHC). CBFb-SMMHC was shown to displace RUNX1 from a newly determined CBF site in the promoter of p15INK4B.