論文の公開元へStructurally-discovered KLF4 variants accelerate and stabilize reprogramming to pluripotency
概要
Cellular reprogramming technology enables to overturn a particular somatic cell type into another desired cell type through the man-made introduction of defined transcription factors that regulate cell differentiation patterns [68, 69]. Pluripotent reprogramming sets up a platform for bioengineering mammalian tissues and organs [98]. Man-made induced pluripotent stem cells (iPSCs) retain the distinguished features of embryonic stem cells, that is to retain one’s own pluripotency and self-regeneration ability in a limitless way in prospect becoming over 200 mammalian adult cell types [101]. The limitless self-regeneration plus the retainment of an undifferentiated, pluripotent state marks both embryonic stem and induced pluripotent stem cells as primary building blocks. Creation of iPSCs suggests an opportunity to replace malfunctioning tissues or/and organs in clinics as well as to establish malfunctioning or physiological tissues and organs for pharmaceutical research or other bio- medical research fields. The theoretical broad-scale applicability is constrained by current low and time-consuming yield of the induced pluripotent cells from primary cell sources.
Given the prospect of iPSCs-based bioengineering and regenerative medicine, industrially compatible generation of invariably high-quality iPSCs is sought after by many research groups. Among cellular reprogramming techniques, the direct lineage reprogramming, performed by activating pluripotency-related genes via viral vector mediated overexpression of selected transcription factors (OCT4, SOX2, C-MYC, and KLF4) is a canon strategy for iPSCs generation [98]. Induced reprogramming by co-expression of transcription factors displays spontaneous potentials and low reprogramming success rates. Estimated causes for faulty reprogramming may be attributed to transcription factors capacities suitable for carrying out physiological tasks only [94, 103].
Transcription factors are generally short-lived proteins governing cellular identity and integrity. By altering gene expression patterns they guide cell state transitions enabling cellular reprogramming. The well-established iPSCs induction protocols use natural transcription factors that fail to meet high-throughput production requirements for cell therapies with the induction efficiency ranging from 0.001 to 0.01 percent [1]. While the addition of certain chemicals or the change of delivery system can increase iPSC colony- forming efficiency, approaches to enforce and adapt reprogramming toolkit through mutation analysis of TFs is majorly unexplored [100, 102]. Limited capacities of natural reprogramming factors might restrain scaling up the successful and synchronous reprogramming into iPSCs.
Among pluripotency reprogramming factors, a zinc-finger transcription factor KLF4 displays a two-faced behaviour attributed to its tilt of being either an oncogene or a tumor suppressor in a context-dependent manner [26]. Consistent with other family members, KLF4 shows a high conservation in carboxyl-terminal residues (that comprise a three zinc-fingers mini- array), as opposed to minimal conservation in amino-terminal residues (that constitute a transactivation domain). The KLF4, being both transcriptional activator and/or repressor, balance multiplex cellular states through modulating cell proliferation, self-renewal, differentiation, pluripotency state, cell survival, apoptosis. Conforming to tumor suppressor activities in many instances, KLF4 is validated to modulate a cell growth arrest reactions’ cascade in a response to DNA damage (observed in intestinal adenomas, colorectal, bladder cancers) [22-23]. Conversely, KLF4 in an assembly with OCT4, SOX2 and C-MYC, transforms terminally differentiated cell phenotype into pluripotent state, eventually resulting in an unlimited proliferation potency [68]. These physiologic effects of KLF4 point out its key role in cell homeostasis and cell cycle integration. Although the scope of carried by KLF4 tasks is well-validated, only a rough estimate of how biochemical and structural properties of KLF4 make out (rule out) its mechanisms is described. The ins and outs of KLF4 functionality remain poorly understood in overall [21].
KLF proteins are necessary intermediate in processes of prevention malignant transformation and maintenance of stem cells viability. The characteristic unit of KLFs is a carboxy-terminal array of zinc fingers, an evolutionary conserved DNA-binding domain that facilitates binding to closed chromatin and recruitment of protein-protein interactions, co- activator enzymes. Compared to other DNA-binding proteins, KLFs seem to use a single discrete zinc finger domain for their three-dimensional allocation and sequence specific DNA-binding rather than a full-length stretch of a protein [28, 29].
Despite binding to identical cis-regulatory DNA sequences (elements), KLF family members display contrasting transcriptional activities in the context of cell cycle regulation. For instance, anti-proliferative activity of Klf4 antagonizes Klf5, which stimulates proliferation, in intestinal and skin epithelial cells. On the other side, Klf4 shares an overlapping (mutually exclusive) activities with Klf5, Klf2 and Klf17 in maintaining pluripotency and self-renewal of mouse embryonic stem cells [20]. Congruent with its role in sustaining pluripotency state, Klf4 plays a central role in iPSCs technology by inducing generation of pluripotent stem cells, an ability attributed only to a specific representatives of transcription factors, namely pioneer transcription factors [104].
Transcription factors exert gene regulation functions through the direct DNA binding, therefore residues constituting a DNA-binding domain are believed to underline functional specificity. One-to-one hydrogen bonds in amino acid-base interactions are viewed as major constitutors of transcription factor-DNA complex stability and reciprocal affinity, especially in the factors of sequence-specific DNA readout, such as pioneer transcription factors that regulate milestone events of cell state transition gene networks. Compared to the unspecific readout mechanism, the indiscriminate specificity towards DNA bases that is preferred by histone proteins involved in broad-base DNA interaction, a highly specific DNA readout is essential for the correct functioning of pioneer TFs that drive remarkable phenotype- changing transitions and maintain a particular cell state.
Here, determination of the several deoxyribonucleic (DNA) bases bonded-amino acid residues of a zinc finger structural class transcription factor, namely KLF4, followed by biophysical, biochemical, computational and bio functional in vitro analyses, suggested differential spatial-temporal conformation and genome-wide binding pattern of a particular residue substituted KLF4 variant that displayed remarkable iPSCs generation activities, exemplified by cell yield acceleration and yield purification for the high-quality homogenous population. Using cell culture experiments functionally non-redundant residues of a zinc finger domain were sorted out and were defined by complete loss or significant impairment of pluripotent function; they represented a major portion of the profiled residues. In vitro screening has also identified a single variant with an advanced reprogramming activity.
Using the methods of cell culturing, conducted in both mouse and human cell lines, successfully reprogrammed colony numbers were calculated (GFP Nanog Fluorescence, immunocytochemistry for Nanog) in a time-course manner indicating that L507A variant urged both faster and greater induced pluripotent stem cells compared to the WT KLF4. To further weight the importance of protein L507 site in pluripotency induction, all the natural amino acid residues were probed, uncovering a stronger favorability of smaller molecular weight (roughly proportional to the size) amino acids to better reprogramming outcomes.
Integrating ChIP-Seq and RNA-Seq data from the same cell samples revealed that KLF4 L507A variant featured enhanced transcription rates of a pluripotency-related genes, including Dppa5a (Esg1), Dsg2 (Desmoglein-2), and Klf5, that were deemed specific for the higher reprogramming efficiency. To introduce the structural reference to biological experiments’ calculations, the atomic level details of DNA-protein complex that show a formation of new direct linear bonds and a presence of a new protein conformation with nearly 33% retention rate has been provided by Molecular Dynamics simulations.
With the provided empirical evidence this study exemplifies that a particular amino acid alteration turns on a prominent practicality in an otherwise unsatisfactory natural TF. Thus, TF-based iPSC conversions utilizing remodeled TFs enable high fold change cost-efficient raise in mouse and human iPSCs generation efficiency, having only minimal alteration in the natural TF analogues.
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