“How fleeting are the wishes and efforts of man! How short his time, and consequently how poor will be his results, compared with those accumulated by Nature during whole geological periods!”

Charles Darwin said as he marveled at what natural selection could potentially do given vast periods of time. Darwin could not quantify how much time could some beneficial trait take to appear and become fixed in the population, therefore he assumed that the millions of years would be sufficient to produce anything. Fortunately mathematicians have not simply assumed this but have tried to quantify how long could we expect a certain trait to appear and become fixed in the population.

Evolutionary change begins with changes in DNA caused by random mutations, these changes can be in existing genes or in regions of DNA which regulate the expression of genes. In the previous post, I surveyed work done on calculating the time it takes for a particular DNA regulatory sequence (promoter) to appear in a section of DNA. The survey revealed that in the human species a 6 letter promoter could take billions of years to appear in some individual of the human race. In order to reduce the time it takes for a promoter to arise – the scientists claim that promoters/transcription factors (TF’s) can arise anywhere in DNA and still confer some selective advantage. This means that TFs should be general and can be placed at any position in DNA and still function. It is this claim which I evaluate in the following survey and come to the conclusion that TFs are not general but specific – in other words life is context.

A recap on what are transcription factors?

Transcription factors are proteins involved in the process of converting, or transcribing, DNA into RNA. Transcription factors include a wide number of proteins, excluding RNA polymerase that initiate and regulate the transcription of genes. One distinct feature of transcription factors is that they have DNA-binding domains that give them the ability to bind to specific sequences of DNA called enhancer or promoter sequences. Some transcription factors bind to a DNA promoter sequence near the transcription start site and help form the transcription initiation complex. Other transcription factors bind to regulatory sequences, such as enhancer sequences, and can either stimulate or repress transcription of the related gene. These regulatory sequences can be thousands of base pairs upstream or downstream from the gene being transcribed. Regulation of transcription is the most common form of gene control. The action of transcription factors allows for unique expression of each gene in different cell types and during development.

Transcription factors are proteins that bind to DNA in order to regulate the expression of a certain gene.

1. Transcription factors are specific and work within highly specific contexts

“Enhancers are essentially developmental transcriptional regulatory modules, in that they contain binding sites for multiple transcription factors (TFs) that function during development and differentiation (3). Although it is likely that housekeeping genes can carry distant regulatory modules in addition to promoters, enhancers are mainly dedicated to controlling tissue-specific gene expression (45, 124), allowing for quantitative tuning and timely regulation of transcription.”[i]

Each gene must be produced at the right time and in the right quantity in the specific cell required. Specific transcription factors control the transcription of specific genes and therefore recognize specific DNA sequences to allow binding. It means not any transcription factor will be able to be functional at any promoter section of DNA. Specific cell types and tissues require specific transcription factors to control their specific proteins

“In the developing spinal cord, bHLH transcription factors are expressed in a number of discrete domains along the dorsoventral axis of the neural tube. Some of these bHLH transcription factors, such as the Drosophila biparous/tap-related genes Neurogenins (Ngns) and the atonal-related gene Math1, show expression patterns restricted to specific progenitor domains of the spinal cord”[ii]

Certain combinations of transcription factors are specific to cell types and even particular locations of certain organs. For examples there are different neuron cells which require different combinations of transcription factors that determine their cell specificity. A different transcription factor would lead to a different cell type thereby preventing proper development of the spinal cord for example. This means that DNA sequences are highly specific to transcription factors for and are not general.

“One of the key discoveries during the past decade is that unique combinatorial arrays of LIM-HD transcription factors, the LIM code, define the target specificity of individual motor neuron subtypes.”[iii]

LIM-HID transcription factors determine the target specificity of individual motor neuron subtypes. It means substituting LIM-HD transcription factors with any other transcriptional factor would disrupt the target specificity of motor neuron subtypes.

“Although context- independent binding (i.e., binding events shared across multiple conditions) is common [162,199,204], context-specific binding is substantial in all cases, suggesting that regulatory specificity is often achieved at the level of TF–DNA binding. Importantly, DNA accessibility is dynamic, with important differences in accessibility across cell types or developmental stages within a cell type [143,209–211]. Thus, the chromatin environment is modified by cellular context, likely through the pioneer TFs expressed in a given context, which, in turn, can impact the binding patterns of nonpioneer TFs”[iv]

2. DNA sequences are highly specified requiring a full match for the transcription factor to bind to it

“A similar transcriptional activation effect of DNA binding has been demonstrated for the Fushi-tarazu (Ftz) protein. This protein binds specifically to the sequence TCAATTAAATGA. As with Ubx, linkage of this sequence to a marker gene confers responsivity to activation by Ftz, such activation being dependent upon binding of Ftz to its target sequence, a lone base pair change which abolishes binding, also abolishing the induction of transcription (Fig. 4.10).”[v]

Proteins bind to specific DNA sequences and changes in one nucleotide can prevent the protein from binding and therefore prevent the correct function of the gene. It means that when a transcription factor evolves all the target letters would have be matched. Recall that Durett had found long waiting times for a full match for a DNA TF site and assumed that a full match is not required for the function to occur. However a full match is required which means it would take an unreasonably long time for a functional transcriptional factor site to arise and become fixed in the population.

3. Transcription factors are interdependent and can require several other specific transcription factors to produce a specific biological function

“In addition, sequence information is often an insufficient predictor of TF binding because in vivo TF binding preferences are influenced by additional variables, including interaction with cofactors and chromatin accessibility.”[vi]

Transcription factors often require cofactors to be able to regulate properly specific genes. Therefore evolving a transcription factor (TF) that requires a certain cofactor (a specific protein able to bind to the specific TF) will require a large amount of time. It also means replacing a specific transcription factor that functions in conjunction with other specific cofactors with some other different transcription factor will disrupt the function of those cofactors. Recall cofactors bind to specific parts of a transcription factor and substituting the TF with a different one means the cofactors will not be able to bind to it. Proteins have specific structures and binding sites for specific substrates.

“Interestingly, structural studies have shown that Ubx and Exd bind to opposite sides of the DNA and that a short region of Ubx N-terminal to the homeodomain extends round the DNA and inserts into a cleft in the Exd homeodomain resulting in interaction of the proteins and enhanced DNA binding by the complex”[vii]

The interaction is facilitated by the specific protein shapes and structures of the respective proteins, hence changing the Exd binding site to Antp binding site will result in a loss of gene transcription because the Ubx and Antp structures are incompatible. Therefore binding sites are not general but specific especially when they work in collaboration with other protein transcription factors.

• Not any transcription factor will work in conjunction with another.

“many TFs do not bind DNA as single entities but, rather, in the form of obligate heterodimers such as TFs containing bZIP, bHLH, MADS box, or Rel DNA binding domains. Since the focus of DNA binding specificity determination studies has largely been on single protein-DNA interactions, DNA binding motifs for such heterodimers are underrepresented in current regulatory lexicons”[viii]

• The majority of transcription factors do not function alone but rather with other transcription factors to form a multi protein complex.

4. Transcription factors and genes form highly specific gene regulatory networks

Genes work in context with other genes in gene regulatory networks.

Figure 4: Gene hierarchy levels. Source: Gene Regulation: Gene Control Network in Development. Smadar Ben-Tabou de-Leon and Eric H. Davidson.[ix]

Genes are expressed in specific contexts determined by gene regulatory networks (GRN). Figure 1 illustrates how GRNs function. Gene B for example is expressed when activated by Gene A. It then activates Gene C which further enhances expression of Gene B and activated Gene D producing some specific biological output. Simply changing the transcription factor of Gene B to another one would mean the (1) whole network loses function; (2) Gene B is activated at the wrong time thereby disrupting the GRN.

Figure 5 – β-catenin-wnt8-blimp1 subcircuit (a) Left: An illustration of the β-catenin-wnt8-blimp1 subcircuit in two neighboring cells. In the two cells activated Disheveled blocks GSK-3 from degrading β-catenin. Stabilized β-catenin enters the nucleus and forms a permissive complex with TCF1 that turns on blimp1. Blimp1 and β-catenin-TCF1 complex together activate the expression of wnt8, a signaling molecule. Wnt8 reception by the Frizzled receptor of the neighboring cell further activates the Disheveled protein in a community effect. The subcircuit is turned off by Blimp1 autorepression and the successive shutoff of the wnt8 gene. The reception of the Wnt8 signal by the next tier of cells turns on this subcircuit there, so the expression of wnt8 and blimp1 forms a ring pattern that advances from the vegetal plate toward the animal pole ( J. Smith & E.H. Davidson, unpublished data)[x]

• The network illustrates how simply changing the Blimp1 gene to be activated by another transcription factor would disrupt the expression of the wnt8 gene. Coordinated changes to how genes are expressed are required to make functional changes to gene regulatory networks.

Davidson who has pioneered work on development gene regulatory networks confirms that removing one gene in a network leads to the collapse of all the entire network, he says:

“…some aspects of dGRN structure appear much more impervious to change than others. For example, a frequently encountered type of subcircuit in upstream regions of dGRNs consists of two or three genes locked together by feedback inputs (Davidson, 2010). These feedback structures act to stabilize regulatory states, and there is a high penalty to change, in that interference with the dynamic expression of any one of the genes causes the collapse of expression of all, and the total loss from the system of their contributions to the regulatory state”

Summary of major points

1. Transcription factors are highly specific and work within highly specific contexts.
2. DNA sequences are highly specified and require a full match for a transcription factor to bind to the sequence.
3. Transcription factors are interdependent and often work in context with other specific transcription factors to achieve some biological function.
4. Transcription factors proteins along with the genes they regulate for complex gene regulatory networks which require all parts of the network present to achieve some specific biological function.

Why does this matter?

Recall that changes in how genes are expressed and controlled is considered to be one of the important ways in which evolution occurs.

1. Mathematical calculations based on population genetics models for human populations for example, show that waiting time for mutations in regulatory DNA regions that will cause different transcription factor proteins to bind there takes very long time. Waiting for a specific 10 DNA letter regulatory change can take up to billions of years. This directly contradicts common descent which claims that humans diverged from their common ancestor with chimps 6 million years ago.
2. In order to preserve neo darwinian theory – the evolutionists then assume that transcription factors do not have to specific but rather general. This reduces the waiting time considerably. However it assumes that any transcription factor can be placed at any place in the human genome and still confer a function.
3. As the survey above illustrates – this is certainly false. Transcription factors work in highly specific contexts and cannot be simply substituted and placed anywhere in the genome.
4. Therefore random mutations and natural selection are not effective mechanisms for explaining changes in regulatory genes. Therefore the surveys, Evolution’s time problem – waiting for genetic change; Evolution’s time problem – new ways of regulating existing genes; show that evolution does not have an adequate mechanism for generating new protein coding genes and new regulatory genes.

Bibliography

[i] Gioacchino Natoli1 and Jean-Christophe Andrau. Noncoding Transcription at Enhancers: General Principles and Functional Models. Annu. Rev. Genet. 2012. 46:1–19. 10.1146/annurev-genet-110711-155459

[ii] Ryuichi Shirasaki and Samuel L. Pfaff. TRANSCRIPTIONAL CODES AND THE CONTROL OF NEURONAL IDENTITY. Annu. Rev. Neurosci. 2002. 25:251–81. doi: 10.1146/annurev.neuro.25.112701.142916

[iii] Ibid

[iv] Matthew Slattery, Tianyin Zhou, Lin Yang, Ana Carolina Dantas Machado, Raluca Gorda, and Remo Rohs. Absence of a simple code: how transcription factors read the genome. Trends in Biochemical Sciences, September 2014, Vol. 39, No. 9.

[v] David S. Latchma. FAMILIES OF DNA BINDING TRANSCRIPTION FACTORS

[vi] Matthew Slattery, Tianyin Zhou, Lin Yang, Ana Carolina Dantas Machado, Raluca Gorda, and Remo Rohs. Absence of a simple code: how transcription factors read the genome. Trends in Biochemical Sciences, September 2014, Vol. 39, No. 9

[vii] David S. Latchma. FAMI L IES OF DNA BINDING TRANSCRIPTION FACTORS

[viii] Bart Deplancke, Daniel Alpern, and Vincent Gardeux. The Genetics of Transcription Factor DNA Binding Variation. Cell 166, July 28, 2016

[ix] Smadar Ben-Tabou de-Leon and Eric H. Davidson. Gene Regulation: Gene Control Network in Development. Annu. Rev. Biophys. Biomol. Struct. 2007. 36:191–212

[x] Michael Levine and Eric H. Davidson. Gene regulatory networks for development. PNAS April 5, 2005 vol. 102 no. 14 4936-4942.