Transcription

Congenital Heart Disease: Mutations of Transcription Factors

Normal Heart Development

The progenitor cells for mammalian heart development are located in the anterior lateral plate mesoderm. At about day 15 of human embryonic development, the progenitor cells condense into two lateral heart primordia. These include lineage precursors for the myocardial and endocardial lineages. At 3 weeks of human development, the cardiac precursors move toward the center, forming a primitive linear heart tube. This loops to the right when cells from the second heart field are added to the inflow and outflow locations. Next, within the outflow tract (OFT), endocardial cushions (subset of cells involved in septation) begin to be formed and they are also formed at the common atrioventricular canal during the sixth and seventh week of development. These cushions aid in the separation of the heart into four chambers and divide the OFT into the aorta and pulmonary artery. At this time the early conduction system begins developing and contributions from the neural crest and proepicardium (progenitor cells near the venous pole) occur. Later on, extensive remodeling of the heart occurs prior to assuming the mature four-chambered structure with divided inflow and outflow. The developing heart also forms valve leaflets and a functional conduction network. The development of the human heart is summarized in Fig. 12.1 .

Transcription Factors Control Heart Development

Normal development of the heart is dependent on the functions of several transcription factors. The central group of factors involves the GATA family of zinc-finger proteins (GATA4, 5, and 6). Others are the MADS box proteins (the term, MADS, is derived from the first letter of four original members of this group: MCM1, AG, DEFA, and SRF). These proteins recruit other transcription factors into regulatory complexes. Other core factors in heart development are T-box factors (Tbx1, Tbx2, Tbx3, Tbx5, Tbx18, and Tbx20). Isl1 [lim-homeodomain (HD) protein] also is essential. These factors interact with themselves and other transcription factors to regulate the maturation of the cardiac chambers. They are also critical for the development of the conduction system and for remodeling of the endocardial cushion. Of these factors the most studied are Nkx2–5, GATA4, and Tbx5; these factors are critical for the development of the heart. Congenital heart disease is associated with mutations in the genes for these factors. Fig. 12.2A and B shows transcription factors and their interactions that are involved in myocardial development and heart morphogenesis.

GATA4

GATA4 is a zinc-finger DNA-binding protein of 442 amino acids and a molecular weight of 44,580 Da. Two types of GATA4 are differentiated based on the position of the zinc finger: either at position 217–241 or at 271–295. It is a transactivation factor that binds to the sequence 5′-AGATAG-3′. GATA4 plays a key role in the development of the heart. It is involved in the induction of cardiac-specific gene expression mediated by bone morphogenetic protein (BMP) through its binding to the BMP response element DNA sequences within cardiac activating domains. In cooperation with another transcription factor, NKX2.5 (or Nkx2–5), it promotes cardiac myocyte enlargement. GATA4 has several biological functions, but a major one is the morphogenesis of the atrial septum, including the development of the arterioventricular canal and the associated valve formation.

Through gene mutation, there are nine positions in the GATA4 protein molecule where point mutations occur involving the change in a single amino acid. These cause ventricular septal defects (VSDs) resulting in abnormal communication between the lower two chambers of the heart. VSD may occur alone or in combination with other cardiac malfunctions. If these defects go unrepaired, they can result in enlargement of the heart, congestive heart failure, pulmonary hypertension, arrhythmias, and possibly sudden cardiac death. A schematic of the GATA4 protein showing its functional domains and the locations of mutations and phenotypes of congenital heart defects is in Fig. 12.3 .

GATA4 is also phosphorylated at position 105, a serine residue, in the sequence PPV S PRFSF by mitogen-activated protein kinase (MAPK) (also known as extracellular signal-regulated kinase). The phosphorylated form is apparently more active in binding to DNA.

Nkx2.5

NKX2.5 is a homeobox protein of 324 amino acids and a molecular weight of 34,918 Da that is involved in the differentiation of the myocardial lineage. It is a transcriptional activator of the atrial natriuretic factor in cooperation with GATA4. It is under transcriptional control by PBX1 ( P re B -cell leukemia homeobo x 1). The domains of the NKX2.5 protein are shown in Fig. 12.4 .

A point mutation within the homeobox domain occurs at amino acid 145 where phenylalanine is converted to a serine (F145S). The homeobox is a DNA-binding domain (DBD) first discovered in Drosophila . It is a sequence of about 180 base pairs giving rise to about 60 amino acids. The homeobox domain occurs in proteins that are transcription factors which are involved in the patterns of anatomical development. The HDs of transcription factors have a characteristic DNA-binding fold. The crystal structure of NKX2.5 is known and it binds to two DNA sequence motifs TGAAGTG/TCAAGAG, straddling them both at the same time ( Fig. 12.5 ).

The NKX2.5 is apparently controlled by phosphorylation–dephosphorylation. The protein occurs both in the cytoplasm and in the nucleus, and its subcellular location is probably driven by its phosphorylation status.

Tbx5

Tbx5 (T-box transcription factor 5) is a protein of 518 amino acids and a molecular weight of 57,711 Da. It is found in the cell nucleus but there are also reports of its location in the cytosol, cytoskeleton, and Golgi apparatus. It is involved in the transcriptional regulation of genes specifying mesoderm differentiation, especially in heart development and in the differentiation of cardiac progenitors. Mutations in the gene for this transcription factor are associated with the Holt–Oram syndrome that is a developmental disorder affecting the heart and upper limbs. Pictured in Fig. 12.6 is the gene for Tbx5 (A) and the mutations in the Tbx5 protein that cause the Holt–Oram syndrome (B).

Aside from the errors in the formation of the upper limbs, about 75% of persons with the Holt–Oram syndrome have potentially life-threatening cardiac problems. Usually, there is a defect (hole) in the septum separating the right and left sides of the heart. If the hole occurs in the septum between the upper chambers of the heart (atria), it is an atrial septal defect. If it occurs in the septum between the lower chambers of the heart (ventricles), it is a VSD. In addition, some patients have conduction disease involving abnormalities in the heart electrical system that can lead to bradycardia (slow heart rate) or fibrillation (uncoordinated heart rate). The Holt–Oram syndrome is autosomal dominant, meaning that only one copy of the altered gene is enough to cause the disease. The syndrome occurs in 1 of 100,000 individuals.

Transcription Factors Involved in Cardiac Hypertrophy

Hypertrophy of the heart is the abnormal enlargement of the heart muscle that results from increases of the size of the cardiac myocytes as well as changes in other components, such as the extracellular matrix. The condition results from biomechanical stress (including hypertension) that can progress to heart failure or even sudden death. It is a maladaptive process resulting from the hypertrophic signaling cascade of which transcriptional factors are prominent as capitulated in Fig. 12.7 .

Transcription Factors and the Transcription Complex

Transcription is the synthesis of RNA [messenger RNA (mRNA)] from DNA (gene) template. Initiation, elongation, and termination are the phases of transcription . Double-stranded DNA must be opened to enable RNA polymerase to bind to the gene promoter (the regulatory region usually upstream from the gene). The transcriptional apparatus is complex and requires that transcription factor proteins bind to DNA or associate with other proteins, including RNA polymerase , at the transcription site. A cartoon of the transcriptional complex is shown in Fig. 12.8 .

While repressors ( purple oblong in Fig. 12.8 ) can bind to silencer sequences in DNA that can be located very far upstream from the core promoter, enhancer DNA sequences that also may be located very far upstream (thousands of bases) from the core promoter form complexes with sequence-specific transcription factors, called enhancer-binding proteins , such as transcription factor IIB (TFIIB) and TFIIA that help to form the transcription complex, bringing the sites into direct contact, and increase the rate of transcription. Another example would be the proteins that bind to the CCAT box.

A molecule of RNA polymerase II ( pol II ) binds to the transcriptional start site. Pol II is a complex of 10 different proteins ( Fig. 12.9 ).

A simplified model of RNA pol II in a transcriptional complex is shown in Fig. 12.10 .

The start site is at the beginning of the information encoding mRNA. The template strand of DNA is the noncoding strand that is used for the formation of mRNA that becomes a copy of the coding strand of DNA, except that thymine (T) is replaced by uracil (U). Pol II (about 35 base pairs upstream from the start site) lies across the template strand of DNA in the 3′–5′ direction, and the mRNA produced is an exact copy of the coding DNA strand in the 5′–3′ direction. Transcribing a specific gene requires a specific array of regulatory factors, which allows the transcription of one gene without turning on the expression of other genes that require a different array of transcription factors. In many cases a group of genes will be expressed by the same transcription factors . As mentioned previously, the process of transcription involves three phases: initiation , elongation , and termination . Initiation is the binding of RNA polymerase to the double-stranded DNA molecule. The core transcriptional elements are the TFIIB recognition element ( BRE ), the TATA box , the initiator element ( INR ), and the downstream promoter element as schematically shown in Fig. 12.11 .

In the figure the sequences defining each motif are shown. The BRE has the sequence G/C,G/C,G/A,CGCC. Where the 3′ conclusion of BRE is reached, the 5′ of the TATA box starts. Sometimes, at a position upstream, a CCAAT box can be found close to the initiator, INR. The CCAAT motif is often found when a TATA box is absent (the TATA box occurs in about 25% of human gene promoters); the CCAAT box specifies the binding of nuclear factor-1 and has the sequence: 5′-GGCCAACTC-3′. A CCAAT box is found in about half of the vertebrate promoters.

The TATA-binding protein is required by all RNA polymerases (pol I, pol II, and pol III). The TATA box is about 25–30 base pairs upstream from the start site near the INR. However, transcription can occur when the TATA box is absent from the promoter. The transcriptional preinitiation complex is made up of transcription factors, including transcription factor IID (TFIID) during initiation, the TATA-binding protein, and RNA pol II bound to the promoter. The TATA-binding protein transcriptional activator factors ( TAF s) include the TATA-binding protein, TFIIB, TFIIE, TFIIF, and TFIIH ( Fig. 12.12 ).

To permit the binding of RNA pol II and the process of initiation, other factors play a role as shown in Fig. 12.13 .

RNA pol II is a complex enzyme consisting of 12 subunits ( Rpb 1 through 12). The subunits 1, 2, 3, 5, 6, 8, 10, 11, and 12 are conserved in RNA pols I, II, and III. The total mass of all 12 subunits is 513.6 kDa.

The bending of DNA occurs when the TATA-binding protein binds to the TATA box . This provides a saddle structure for other transcription factors to form a complex ( Fig. 12.12 ). TAFs involve histone acetyltransferases (HATs) , protein kinases , coactivators , and other activities. The INR interacts with the TATA-binding protein and with the coactivator, SP1 (spacing relative to TATA-binding protein), and the largest subunit of RNA pol II. The sequence of the INR is YYA ₊₁ NT/AYY where Y is a pyrimidine and A is the transcriptional start site. The transcriptional start site begins with the start codon (usually AUG ; specifies methionine) one position after the 5′-untranslated region, the 3′-end of the leader sequence . The leader sequence often includes information for the destiny of the translated protein but is not part of the coding region.

RNA pol II binds directly to the INR box and initiation occurs when the TATA-binding protein and the TAFs dissociate from RNA pol II, and RNA pol II begins a forward movement over the opened strand of DNA (strand opening by helicase and ATPase ). Some of the transcription factors are dissociated, but TFIIB remains at the initiation site with other transcription factors. During elongation, RNA pol II uses a clamp mechanism to move forward. It also has a positively charged saddle structure for the binding of DNA and RNA. Three zinc ions stabilize the fold of the clamp that closes on DNA and RNA to trap the DNA template and the RNA transcript ( Fig. 12.14 ). A molecular view of RNA polymerase is shown in Fig. 12.15 .

The active site that includes the hybrid of DNA and the transcribed RNA being formed has a preference for ribose nucleotide triphosphates over deoxyribonucleotide triphosphates as would be expected for the growing RNA. In Fig. 12.16 is shown the addition of a ribonucleotide triphosphate to the growing mRNA chain in the RNA pol II overall reaction.