Control Points For Gene Expression

All of the stages of gene expression are points at which regulation can be exerted. However, the primary point of control is at the level of transcription. Many promoters and enhancers of myocardial genes have been cloned and transcription factors

Table 28.1 Some examples of key transcription factors expressed in the heart

Factor

Gene family

DNA binding site

Expression pattern

Examples of gene regulated

Myocardin6

HIF-1 9

MADS box family MADS box family SAP domain family

GATA family Homeobox bHLH

CTA(A/T)4TAG CC(A/T)6GG ("AcrG box") Does not bind directly to DNA, but binds to SRF as co-factor

VtGATA79

TNNAGTG (high affinity) C(a/t)TTAATTN (lowaffinity) a/p heterodimers bind CANNTG

Widely expressed Widely expressed Heart

Myocardium, endoderm Cardiac mesoderm

Ubiquitous; HIF-1 ß constitutively expressed, changes in levels of active HIF-1 a induced by hypoxia

TnIc CK-M

a-cardiac actin, SM22, c-fos SM 22, ANF

a-MHC, cardiac TnC, TnIc MLC2v, ANF, cardiac a-actin

MLC2v, ANF, cardiac a-actin a-MHC, cardiac a myosin heavy chain; ANF, atrial natriuretic factor; bHLH, basic helix-loop-helix; CK-M, muscle creatine kinase; HIF-1, hypoxia inducible factor-1; MEF-2, myocyte enhancer factor-2; MLC2v, ventricular myosin light chain 2; SM22, smooth muscle 22; SRF, serum response factor; TnIc, cardiac troponin I; TnC, troponin C.

active in heart and belonging to a variety of gene families have been identified in recent years (table 28.1).4-9 The overwhelming observation that can be drawn is that expression of individual genes is regulated (1) through the coordinate binding of different types of transcription factors, (2) by interactions between factors and with ancillary co-factors such as histone acetylases (HATs) or deacetylases (HDACs), which do not necessarily bind to DNA themselves, and (3) through signal transduction pathways which influence their activity by, for example, phosphorylation.3 Most transcription factors are modular in structure, containing separable protein domains that carry out a particular function such as DNA binding, dimerisation with other family members (for example, the related bHLH proteins HIF-1a and P, products of separate genes9) or serving as transcriptional activation domains (TADs) to promote high level transcription. Ultimately, once bound to DNA transcription factors interact with other bound proteins and act to increase the rate of RNA synthesis. TAD activity, which is often measured by introducing cloned transcription factors into cells grown in culture (see below) may be intrinsic or may reflect the binding of a co-activator or co-factor protein which itself possesses significant activation properties. For example, myocardin is a recently identified heart restricted co-factor for the ubiquitously expressed serum response factor (SRF).6 SRF binds to the promoters of several genes expressed in heart, including cardiac a actin, and has been shown to interact with many factors including the homeobox factor Nkx-2.5 and the zinc finger factor GATA-4 to regulate expression. In contrast to Nkx-2.5 and GATA-4, which are transcriptional activators in their own right, myocardin does not bind to DNA but complexes with bound SRF and serves as an extremely potent co-factor for transcription, promoting up to a thousand-fold activation in combination with SRF.

Transcription factors may be expressed in a highly tissue restricted manner or at particular developmental stages, in turn regulating the expression of their target genes. For example, GATA factors are expressed from the earliest detectable stages of cardiogenesis and may play a role in gene regulation at this stage.10 Later, Nkx2.5 shows regional variation in expression and may play a specific role in the developing myocardial conduction system. Most transcription factors can be grouped into gene families on the basis of sequence similarity in regions of functional importance, such as a DNA binding domain or a protein dimerisation interface. Such a high degree of sequence homology allows new family members to be discovered by searching DNA or protein sequence databases across diverse phyla. In this way Nkx-2.5 was identified as the mammalian homologue of a Drosophila gene called tinman, (named after one of the characters in The Wizard of Oz) originally identified as a mutation that resulted in lack of development of the heart equivalent in the fly, the dorsal vessel.8 In humans, mutations in the Nkx-2.5 gene have been correlated with a variety of cardiac anomalies including tetralogy of Fallot and idiopathic atrioventricular block.11

Currently, there is renewed interest in the role that chroma-tin structure plays in regulating gene expression.12 It has long been known that when DNA is wrapped around histones in the form of chromatin, gene activity is silenced, and that the localised unwinding of the DNA from chromatin, accompanied with histone displacement, is vital to allow gene expression to progress. Central to recent studies has been the identification and biochemical characterisation of proteins that possess HAT or HDAC activity, adding or removing, respectively, acetyl groups from exposed lysine residues on histones. HAT activity correlates with activation of gene expression, while HDAC activity results in repression.13 In several cases the functions of these proteins have been shown to be intimately linked to the state of transcription factors binding to target genes. For example, the active heterodimeric form of the basic helix-loop-helix transcription factor HIF-1 (table 28.1) senses changes in partial pressure of oxygen and thus acts as a hypoxia sensor in several systems, including angiogenesis and vascular remodelling.9 Once bound to the DNA of target genes for regulation, C terminal TADs in the HIF heterodimer interact with transcriptional co-activators such as the CREB-binding protein, CBP. (In cardiac muscle, the most likely co-activator is the related protein p300). These large proteins possess intrinsic chromatin remodelling activity by recruiting to the DNA still more proteins which allow chromatin to unwind. Probably the best understood system is currently that involving retinoid and steroid hormone receptors such as thyroid hormone receptor a1 (TRa1) that, once it has bound its cognate ligand T3, activates expression of genes such as cardiac a myosin heavy chain.2 The binding of T3 to the hormone binding domain of TRa1 results in a conformational change in the proteins' structure, allowing the receptor to interact with co-activators. The net result is that HAT activity promotes localised unwinding of chromatin, allowing access of RNA pol II and basal transcription factors to the DNA. In the absence of T3, the nuclear receptor still binds to DNA but interacts instead with co-repressors such as N-CoR and SMRT, which then recruit HDACs to the DNA, leading to chromatin condensation and repression of gene expression.14

The activation of transcription factors by phosphorylation is a focal point for transducing extracellular stimuli through

Interaction Between Bacteia And Metal

Figure 28.2 Linking MEF2 to hypertrophy. Hypertrophic stimuli activate intracellular signalling pathways. Upstream protein kinases (for example, MEKKs) activate MAP kinase family members (MKKs) which in turn phosphorylate the three MAP kinases p38-MAPK, JNKs, and ERKs. MAP kinases have been linked to the phosphorylation of certain transcription factors, for example, p38-MAPK has been shown to activate MEF2 family members (see text for details). This implicates MEF2 proteins as direct transducers of intracellular signalling pathways to bring about some, or all, of the changes in gene expression associated with hypertrophy. Figure courtesy of KA Dellow.

Figure 28.2 Linking MEF2 to hypertrophy. Hypertrophic stimuli activate intracellular signalling pathways. Upstream protein kinases (for example, MEKKs) activate MAP kinase family members (MKKs) which in turn phosphorylate the three MAP kinases p38-MAPK, JNKs, and ERKs. MAP kinases have been linked to the phosphorylation of certain transcription factors, for example, p38-MAPK has been shown to activate MEF2 family members (see text for details). This implicates MEF2 proteins as direct transducers of intracellular signalling pathways to bring about some, or all, of the changes in gene expression associated with hypertrophy. Figure courtesy of KA Dellow.

MAP kinase signal transduction pathways. For example, CBP associates only with the phosphorylated form of the CREB protein, a transcription factor that binds the cyclic AMP response element found in many gene promoters. Once bound to CREB, CBP forms protein-protein interactions with the basal transcription factor TFIIB, allowing transcription by RNA pol II to progress. Members of the myocyte enhancer factor-2 (MEF-2) family of transcription factors (table 28.1), which are widely expressed but appear to be enriched and have particular roles in skeletal and cardiac muscle, may regulate changes in gene expression arising from a hypertrophic stimulus. MEF-2 proteins have been implicated as responders to MAP kinases activated by hypertrophic stimuli in cardiac myocytes.15 The hypertrophic agonists endothelin-1 and phenylepherine activate p38 MAPKs in cultured rat neonatal cardiac myocytes16, and in vivo p38 activity increases in aortic banded mice which go on to develop pressure overloaded hypertrophy (Wang and colleagues 1998, cited in Han and Molkentin15). In rat, a similarly induced hypertrophy results in an increase in DNA binding activity of MEF-2.17 As development of hypertrophy is associated with changes in transcription of various myocardial genes which require MEF-2, this suggests that MEF-2 may be a direct target for MAP kinase signalling (fig 28.2).

A second point at which gene expression can be controlled is at the level of mRNA splicing. The primary RNA transcript of many genes may be alternatively spliced in that certain exons may be excluded or included from the transcript to produce the final mRNA. In this way, multiple proteins may be generated from a single gene according to the combination of exon derived RNA segments that are spliced together. The vertebrate tropomyosin (TM) genes are examples of genes expressed in both cardiac and skeletal muscle that are subject to complex patterns of alternative mRNA splicing. For example, two isoforms of the a-TM gene in Xenopus, differ in their inclusion of alternative 3'untranslated region exons and show restricted expression in the embryo.18 The XTMa7 isoform is found in the somites, from which the skeletal muscle develops, whereas the XTMa2 isoform is expressed in both somites and embryonic heart. In the adult, XTMa2, but not XTMa7, is selectively expressed in striated muscle and heart.

Following translation of the mRNA into polypeptide, production of mature active protein may require several steps, each of which is open to regulation. This is well illustrated by the matrix metallproteinases (MMPs) which are believed to play a key role in myocardial remodelling.19 MMPs are produced in an inactive form (as a zymogen) which requires cleavage to produce active enzyme and are further regulated by specific inhibitory molecules (tissue inhibitors of metallo-proteinases or TIMPs). Examining MMP gene expression at the level of RNA is of importance to understanding gene regulation, but may not therefore be a good indicator of MMP enzyme activity. Clearly, it is important to understand the biological system in question before deciding which is the most relevant level of regulation. For the purposes of this review we will be focusing on the initial stage of gene expression, namely transcription, and the methods of examining this as well as monitoring RNA content.

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  • eugenio piazza
    What is the control point that gene expression involve?
    3 years ago

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