Table 76

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Homeodomain TFs (coded for by homeobox genes) have a basic 60 or 61-amino acid helix-turn-helix (bHTH) protein domain that, depending on its amino acid sequence, binds specific DNA sequences; the third helix extends into the major groove of the DNA that it "recognizes." Amino acids in the NH2-terminal portion of the homeodomain also contact the bases in the minor groove of the DNA double helix. A class called POU TFs also has a second DNA-binding domain. Lim and Pax genes comprise two other homeodomain gene classes. The Hox class is the most well-known; they are involved in neural differentiation, anatomic axis specification (e.g., main anterior-posterior, or proximal-distal appendage axes), and head and eye development.

Zinc finger TFs form domains that include loops made by the binding of spaced pairs of cysteine and histidine around a zinc molecule, leaving a projection ("finger"). Between such fingers is a sequence-specific DNA-binding region. Zf genes include the Sox family that bend DNA to assist in the assembly of regulatory protein complexes, sex differentiation, cartilage development, and hormone and retinoic acid signal reception. They are part of a large number of chromatin-associated genes with a High Mobility Group (HMG) box, a DNA-binding domain that recognizes a consensus sequence (A/T)AACAAT. There are many Sox genes, and several classes of HMG genes that are not closely related to this family, which may or may not include a zf domain.

Leucine zipper TFs are formed of two polypeptides joined by regularly spaced leucine residues (the "zipper") adjoining a DNA-binding region of basic (positively charged) amino acids.

Basic helix-loop-helix (bHLH) TFs form dimers in each of two helical domains, that are separated by a loop; DNA binding is by a region of 10-13 basic amino acids at the end of the first helix in each of the polypeptides. The two molecules may be of the same or from a related bHLH gene. bHLH genes are important in a variety of neural and sensory developmental functions, and include the MyoD genes used in muscle development.

MADS box genes regulate many functions but are particularly prominent in flower morphogenesis (the acronym is the initial letter of the first four family members identified). Along with a variety of other functional domains, these genes have a characteristic 57 amino acid DNA binding motif, and a domain for forming homo- or heterodimers.

Other classes of TFs, such as the "paired" group, have one or two DNA binding domains that are variants of these classes. The TF families are old and have accumulated family members through duplication events in long phylogenetic lineages; there are therefore numerous members overall, that vary among species. For example, there are tens of genes in the Hox homeobox superfamily (39 in humans). Typically, representative homologs in the animal TF families are found across the animal world.

(e.g., receptor-ligand binding leading to TF activation and cis-regulation) is generic and development and its evolution are now frequently viewed in terms of regulatory pathways (Carroll et al. 2001; Davidson 2001; Wilkins 2002). A regulatory pathway or circuit goes from inducing signal to TF activation and hence an activated (or repressed) developmentally downstream target gene. Development is hierarchical in that one circuit can activate subsequent circuits—for example, when the first

TF activates a second TF that then activates subsequent genes. A set of genes regulated by the same circuit has been called a battery of genes. Interactions among contemporary circuits or batteries comprise a regulatory network. It might be worth noting that meshwork could be even more evocative, because two-dimensional networks of regulation are connected to each other through time, with some circuits active and others missing at different times, or with a pathway having different uses at different times, and effects feeding back upon themselves, depending (presumably) on what else is active at each time.

An indicator of the basic importance of this kind of genetic logic is that SFs, their receptors, and TF classes are widely represented in vertebrates and invertebrates, sometimes even plants and bacteria. It is possible in many instances to identify specific orthologs among gene family members in very distantly related groups, such as flies and mammals. Homologous interaction pathways are often deeply conserved, such as between a given ligand, its receptor, and/or the TFs it activates. In addition, such conservation may effect similar function (such as activation in neural or gut tissue), revealing surprisingly deep homologies in basic animal or plant functions.

Members of TF families may be linked in chromosomal clusters that have been conserved for long evolutionary time periods, and the conserved linkage arrangement is related to the control of the expression of the genes in the cluster. However, this is not an automatic need for TF regulation, because multiple TFs, including members of the same families as those in clusters, are chromosomally off by themselves.

Regulatory Control Occurs in Different Ways

Some TFs are autoregulatory; that is, once expressed they enhance their own continued transcription—copies of their own RE sequences are located 5' to the TF genes themselves. Just as apoptosis and proteases remove cells or substances in a variety of remodeling or repair contexts, gene expression can also be down-regulated or inhibited.

Inhibition can be achieved in various ways. One way is pretranscriptional, when other regulatory repressor proteins bind areas around a gene to prevent activation by TFs. Chemical modification of the regulatory region has the same effect, preventing access or binding by TFs. The DNA cannot be opened (e.g., histones removed) to permit access by an activating TF. Earlier, we described posttranscrip-tional regulation by RNAi. There is also posttranslational inhibition. For example, bHTH TFs (Table 7-6) bind REs as heterodimers. The TF protein has separate dimerization and DNA binding domains. There are genes that code for truncated proteins that lack the DNA-binding domain; they form normal dimers, but the result cannot bind DNA properly, which prevents or inhibits the expression of the gene a normal dimer would activate. Similarly, there are receptors that lack one of their domains, and hence reside in the membrane where they receive signal, but the receipt is not transmitted internally to the cell (e.g., Kroiher et al. 2001).

Expression of a particular gene or cell-specific set of genes depends on the required set of TFs and/or inhibiting factors being present at the same time in the cell, meaning that the appropriate set of receptors, second messengers, and the like must also be expressed in the cell, along with the TFs themselves. Cells may require more than one signal for a given action to take place and thus all the requisite receptors may need to be triggered; this means that the extracellular as well as the intra-

cellular environments must be appropriately prepared. Thus, itemizing a pathway by itself is somewhat of an illusion of simplicity.

An Embarrassment of Riches, or Neo-victorian Beetle Collections?

A description of the basic logic of signaling and gene-regulation mechanisms, along with a catalog of their major classes, does not do justice to what actually is going on within a cell. The cascades of regulation that bring about even relatively simple aspects of complex traits are themselves complex (e.g., Carroll, Grenier et al. 2001; Davidson 2001) and often involve parallel shunts or redundancy. Regulatory and signaling pathways can be investigated by a host of experimental in vivo and in vitro methods, including cell culture, animal models, and the direct manipulation of individual genes or regulatory sequences. Experiments dissect pathways component by component in a way to reveal their individual effects (under the particular laboratory conditions being used). As shown in Figure 7-3, regulatory pathways can be intricate, and also, the apparent complexity of a pathway can depend on the method used to study it. For example, in part A, the pathway seen from normal development of relatively simple model organisms can seem quite straightforward. In part B of the figure, the results of different experiments, in different cell types under different conditions, in more complex organisms, can make the "same" pathway appear more complex, because the latter approach reveals more of the phenogenetic equivalence (redundancy, interconnectedness) of "the" pathway.

Experiments with individual known pathways can be supplemented with genomic approaches that identify which of the entire set of an organism's genes are used in a given cell type. With the use of various technologies, it is becoming possible to identify and quantify all the genes that are being expressed in a given cell type. This expression profiling can identify, for example, genes whose expression changes quantitatively or qualitatively before and after cells are subjected to nutritional stress or those that are expressed differently between tumor and normal cells from the same tissue (e.g., Eisen et al. 1998;Tamayo et al. 1999). Expression profiles are useful when the situation is relatively simple or well-controlled experimentally, but they can also reveal the quantitative and complex nature of cell behavior. Redundancies and other interactions may be difficult to discern from an expression list (even with quantitative data) alone.

Expression profiles can reveal whether genes with coordinated expression under the tested circumstances are (1) in coregulated chromosomal clusters, (2) coregu-lated members of gene families, or (3) scattered genes coregulated because they have related function (e.g., networks of TFs and downstream expression cascades). Co-regulation within clusters can be achieved in various ways, and genes on different chromosomes that are coordinately expressed can use the same or different regulatory mechanisms. That is, this can be due to similar REs, or different REs whose coordinated usage evolved; in some instances, phenogenetic drift is probably responsible, if different genes have had a similar common expression but have evolved different regulatory mechanisms. The alpha- and beta-globin genes, and different means of coordinate regulation of Hox genes in vertebrates probably represent instances of the former. But the function could have been achieved by recruiting different, otherwise unrelated genes to some new function.

In some ways, we have in recent years been presented with a sudden embarrassment of riches. There is a danger in taking the details too seriously, mistaking

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