Regulation At Transcriptional Level

Published Date: 02 Nov 2017

Regulation of p16 is a complex process, the reason is that the unique nature of INK4b/ARF/INK4a locus. p16, p15, and p14ARF have different independent promoters, so the product proteins have function in different pathways. They independently activated or repressed under different circumstances (Gill and Peters, 2006). Grouping genes in a single chromosomal domain have advantages, they can be regulated as a whole by the same remodeling event(s) (Kim and Sharpless, 2006), so INK4b/ARF/INK4a locus is coordinately regulated. Recently scientist proved that there is two type regulatory mechanism for p16. These are independent and coordinated regulation of p16.

Independent Regulation of p16

In the past decade transcriptional regulation of p16 has been one of the intense study area because of its role in tumor suppression, senescence, and aging. It is found that transcription of p16 is related to various regulatory elements which present in the p16 promoter (Li et al., 2011).

Ets-Binding Site-Mediated Regulation

There is a conserved Ets-binding site in the p16 promoter in position _124 to _85 interval. Ets1 and Ets2 transcription factors that are able to bind to Ets binding site which activate the p16 promoter and trigger the expression level of p16 in human fibroblasts. Ets1 and Ets2 transcription factors can be activated by MAPK-mediated phosphorylation (Ohtani et al., 2001). Id1 is a a helix_loop_ helix (HLH) protein which is a repressor of p16 expression, physical association of Ets2 with Id1 makes incapable the Ets2-mediated transactivation, while in human diploid fibroblasts abnormal expression of oncogenic Ras increase the binding of Ets1 and Ets2 to the p16 promoter (Zheng et al., 2004). The reason is that Ets2 controls the transactivation of p16, Id1 counterpoise the activation of the p16 promoter which is mediated by Ras-Raf-Mek signaling (Ohtani et al., 2001). In young fibroblasts, p16 is expressed at low levels because of a balance between Ets2 and Id1. Abnormal phosphorylation of Ets2 by oncogenic Ras may override this steady state, thus activate the 16 promoter and transcription. During senescence, the Ras-Raf-Mek signaling is reduced and the Ets2 level is low; consequently the increased level of expression of Ets1 and concomitant downregulation of Id1 result in upregulation of p16. The balance between Ets1/2 and Id1 is like a sensor which detects aberrant growth signals (mitogenic stress and oncogenic stress). Current studies demonstrate that ROS (reactive oxygen species) is a contributor to senescence which is attributed to its transactivation of p16 expression by the Ras-Raf-Erk-Ets signaling pathway (Schmid et al., 2007).

E-Box-Mediated Regulation

Id1 may also effect the transactivation of E47. E47 has an HLH domain, like other class I basic helix_loop_helix (bHLH) proteins (also known as E proteins) (Pagliuca et al., 2000). E47 mediates homo- and heterodimerization with other HLH proteins to regulate gene expression. promoter sequence of p16 contains E-box sequences. There are two E-box elements in the p16 promoter, located at positions _349 (CAGGTG) and _615 (CAGGTG) (Zheng et al., 2004). In case of homodimerization or heterodimerization with other E proteins E47 binds to these two E-boxes and activate the transcription of p16 in senescent cells. Id1 can form only a heterodimer with E47, Id1 has a highly conserved HLH domain but lacks the basic DNA binding domain. Id1 binds to E-box elements and inhibits E47-mediated activation of the p16 promoter (Li et al., 2011). In addition, Myc, is an E-box-binding transcription factor, which binds to the promoter and the first intron of p16 and upregulate its expression in human cells (Gill and Peters, 2006).

Sp1-Binding Site-Mediated Regulation.

There is a GC-rich region within the p16 promoter, which contains at least five assumed GC boxes (positions _474 to _447, _462 to _435, _380 to _355, _76 to _49, and _26 to _1), that are a sign of assumed binding target sites for Sp transcription factors which are Sp1, Sp3, and Sp4 (Wu et al., 2007). In the p16 promoter, there exist a positive transcription regulatory element ( in positions _466 to _451) and also called GC box for for Sp1 binding (Wu et al., 2007). The amount of Sp1 protein does not influence the binding of Sp1 to GC box, Sp1 binding is enhanced by increase in Sp1 affinity during cellular senescence. Furthermore, abnormal expression of Sp1 triggers the transcription of p16 in human fibroblasts (Wang et al., 2008). It is suggested that binding of Sp1 to GC box has a possitive influence on p16 transcription. Moreover, there is a coavtivator with histone acetyltransferase activity which cooperates with Sp1, it is called P300/ CBP. They both activate the p16 promoter activity and p16 mRNA expression. The histone acetyltransferase domain of P300 is able to contribute to p16 transcriptional activation by inducing hyperacetylation of histone H4 at the p16 gene (Xue et al., 2004). P300 is Sp1-dependent in p16 transcription, whereas abnormal expression of P300 is able to induce cell cycle arrest by upregulating p16 expression.

HBP1-Binding Site-Mediated Regulation

There is a binding site for the HMG box-containing protein 1 (HBP1) transcription factor at possition _426 to _433 in the p16 promoter. It is a downstream effector in the Ras-Raf-Mek signaling pathway (Li et al., 2010). Binding of HBP1 to the p16 promoter positively regulates the expression of p16 and induce premature senescence in primary cells.

ITSE-Mediated Regulation.

There is a a negative regulatory element in the p16 promoter which is called ITSE (the INK4a transcription silence element) in location _491 to _485 (Wang et al., 2001). In case of deletion of ITSE in young 2BS cells, the p16 promoter activity increase. In addition, ITSE has a binding site for Myb-related protein B (B-MYB), which is a transcription factor play role in the regulation of cell survival, proliferation, and differentiation (Huang et al., 2011)

Ap1 Site-Mediated Regulation

Ap1 is a transcription factor which is a heterpdimeric and homodimeric protein, Ap1 proteins binds to leucine zipper (bZIP) region in DNA sequence. Ap1 composed of Jun proteins (c-Jun, JunB, and JunD), Fos proteins, Jun dimerization partners (JDP1 and JDP2), and related activating transcription factors (ATF2, LRF1/ATF3, and B-ATF) (Li et al., 2011). There are three AP1-like sites in the mouse p16 promoter, which includes TGACTGA at position _1189, TGACTTCA at position _783, and TGACACA at position _484 (Huang et al., 2011). Ap1 transcription factors has different effects on cell proliferation, arrest and apoptosis (Gill and Peters, 2006). For instance ectopic expression of JunB induces high levels of P16 and induces growth arrest in mouse fibroblasts and reduce the level of proliferation in T3 cells (Shaulian and Karin, 2001). Induction of p16 expression is related to transactivation by binding of JunB to the Ap1 sites in the p16 promoter. Additionally JunB plays role as a downregulator of the expression of cyclin D1. Overexpression of JunB in 3T3 cells prevents the kinase activity of CDK4, thus pRb hyperphosphorylation reduces and G1 phase extends.

In an opposite manner, Conversely, c-Jun upregulates cyclin D1 but downregulates P16, so it promotes cell proliferation (Li et al., 2011).

PPRE-Mediated Regulation

PPRE is a peroxisome proliferator response element which is located at position _1023 in the p16 promoter (Gan et al., 2008). Peroxisome proliferator-activated receptor R (PPARR) negatively regulates the cell cycle progression at the G1 to S phase transition by triggering p16 expression in vascular smooth muscle cells (SMC). There is an association between PPARR and Sp1, PPARR binds to the canonical PPRE region and interacts with Sp1 in the proximal Sp1-binding sites of the p16 promoter, therefore enhance the p16 expression (Li et al., 2011).

Regulation Mediated by Unspecified Elements

There are some regulatory elements which are unsipesifc element but they regulate the p16 expression. SWI-SINF (SWItch/Sucrose NonFermentable) (Stern et al., 1984) is a chromatin remodeling complex,which destabilise histone-DNA interactions to regulate access of binding domains to transcription machinery (Betz et al., 2002) and capable of altering the position of nucleosomes along DNA (Whitehouse et al., 2009) SNF5 is a component of the SWI_SNF chromatin remodeling complex. In malignant rhabdoid tumor cells (MRT) re- expression of hSNF5 cause to G1 arrest by triggering the p16 expression and transcriptional repression of cyclins A, D1, and E (Oruetxebarria et al., 2004). The reason is that there is BAF60a subunit in the mammalian SWI_SNF complex which interacts with JunB, and SWI_SNF complex may initiate the p16 promoter, thus hSNF5 may activate p16 transcription.

Additionally there are a few repressors of INK4a/ARF/INK4b expression. For example Polycomb group (PcG) genes (BMI-1, Cbx7, Mel18) which are first identified in Drosophila, have been reported to repress all three genes (p16 INK4a, p15INK4b, and ARF) (Gill et al., 2004; Jacobs et al., 1999). These Polycomb group of silencers (PcG) maintain patterns of developmental gene expression (Orlando et al., 1998; Francis et al., 2001)

In recent studies, there is a newly identified transcription activator which is called BRG1 is a catalytic component of the SWI_SNF complex (Medina et al., 2008; Becker et al., 2009). Whereas BRG1 I not necessary for cell cycle inhibition which is induced by p16, P16 and BRG1 interaction negatively regulates the chromatin remodeling activity of BRG1 (Li et al., 2011) Recently, lymphoidspecific helicase (Lsh) is demonstrated as a a member of the SNF2/helicase familywhich has a role in p16 regulation (Zhou et al., 2009). Overexpression of Lsh in human diploid fibroblasts postpone the cell senescence by silencing p16. This repression cause to Lsh-related deacetylation of histone H3 at the p16 promoter.

Coordinated Regulation of p16, p14ARF, and p15

Despite the fact that through regulation of p16, p14ARF and p15, there are some stimuli which regulate p16 but not p14ARF or p15, these three genes are expressed both in normal tissues and insignificant number of tumor specimens (with whole INK4b/ARF/INK4a locus) (Ortega et al., 2002; Kim and Sharpless, 2006; Collado et al., 2007). In spite of 50 different regulatory domains p16 and p14ARF mRNAs are extremely stable, which is  hypothetically attributed to their shared 30 sequences (Guo et al., 2010). According to these common regulatory features, there might be a mechanisms controlling p16, p14ARF, and p15 simultaneously. This assumption supports that, p14ARF, and p15 are simultaneously downregulated by expression of PcG proteins, such as Bmi1, Cbx7, Ring1b, or Phc2 (Jacobs et al., 1999). PcG proteins are

transcriptional repressors, they recognize histone modifications so transcriptionally silencing chromatin (Gonzalez et al., 2006).

Bmi1 is a potent repressor of of INK4a. Bmi1 was first identified by its ability to cooperate with Myc in the induction of mouse lymphomas (Haupt et al., 1991; Van Lohuizen et al., 1991) In Bmi1 knockouts it is found that Bmi1-deficient mouse embryonic fibroblasts encounter premature senescence and accelerated accumulation of p16, p19ARF, and p15. Besides, abnormal expression of Bmi1 enlarge the age of death of both mouse and human fibroblasts. Presence of the multiprotein complex which contains EZH2 (a histone methytransferase) and (PRC 2) (Polycomb-repressive complex 2) cooperate with the Bmi1-mediated repression of the INK4b/ARF/INK4a locus (Canepa et al., 2007)

Additionally, Cbx7 is one of the Polycomb protein, , localized to nuclear Polycomb bodies and interacts with Ring1 (Bezsonova et al., 2009; Maertens et al., 2009). Cbx7 transcriptionally repress the INK4b/ ARF/INK4a locus independently from Bmi1 and extends the life span of normal human cells and immortalizes mouse fibroblasts (Gill et al., 2004) Recent studies revealed that CDK6 has a physical interaction with Bmi1 in young MEF cells, thus, recruiting PRC 1 and PRC 2 to the INK4b/ARF/INK4a locus and transcriptionally silence the locus as well as its replication during the late S phase (Agherbi et al., 2009).

There is another transcriptional corepressor which is called CtBP (COOHterminal binding protein), it is capable of access the p16 promoter enhance the PcG-based epigenetic histone mark, therefore help p16 silencing by DNA methylation (Mroz et al., 2008). CtBP-mediated repression of p16 can be reduced by increased level of ROS. CtBP has very little influence on the expression of p14ARF, its influence on the expression of p16 or p14ARF is different.