Research in the Slack Lab
December 2005

MicroRNAs and their targets in development and disease

My lab is interested in the mechanisms of developmental timing, focusing on the role of microRNAs, heterochronic genes and circadian timing genes in biological timing and disease.

Development is a four-dimensional process, explicitly controlled by genes, that begins with fertilization and ends with death. We are using the advantages of the model organism C. elegans to find important genes and mechanisms that control developmental timing. A pathway of timing genes has been described by the identification of mutants where cells terminally differentiate at the wrong time relative to in wild-type animals. The temporal patterning pathway that is emerging includes key temporally expressed transcription factors and small, regulatory RNAs called microRNAs, with many of these genes related to human cancer genes. We are using molecular, genetic and genomic approaches to understand the roles of the lin-4 and let-7 microRNAs, the LIN-14, LIN-42 and HBL-1 transcription factors, and the LIN-41 RING finger protein in the control of developmental timing and aging. In addition, since many of the C. elegans heterochronic genes control timing of cell differentiation and are related to human cancer genes, we are examining the role of their human homologues in disease. An emerging theme is of universal patterning mechanisms acting throughout the animal kingdom. We are extrapolating our work to provide an understanding of how organs are specified at the correct time during development of higher animals. Our work should also lead to a better understanding of human cancer since cancer is often caused by the inappropriate adult redeployment of developmental pathways utilized previously in the embryo.

These are very exciting times in the field of post-transcriptional mechanisms of gene regulation. My lab has pioneered various aspects of the microRNA field and continues to make important contributions to their roles and mechanisms. We intend to continue at the forefront of the field, focusing on temporally-regulated miRNAs and their role in development and cancer. Using these miRNAs as models, we hope to discover how miRNAs are expressed, what they regulate, how they function and what roles they play in development and disease.

We are using molecular, genetic, bioinformatic and genomic approaches to understand:

1. the role of microRNAs (miRNAs) like lin-4 and let-7 in control of gene expression and development. lin-4 and let-7 are founding member of a large family of recently discovered miRNAs, which are small, regulatory RNAs that control essential development processes such as cellular differentiation, proliferation, and apoptosis in animals, including humans. In C. elegans, these two miRNAs control the timing of cell differentiation by regulating gene expression in the heterochronic pathway. These RNAs bind to complementary sequences in the 3'UTR of their target gene mRNAs and through a mechanism that we are trying to understand, down-regulate their translation. We have isolated protein factors that bind to miRNAs and hope that their identity will shed light on the miRNA mechanism.

We have described the minimal sequences necessary for miRNA control of target sequences and have used this information to design bioinformatics screens to identify novel miRNA targets, in an effort to understand how these miRNAs control differentiation. Most of the dozen or so targets we have identified encode transcription factors, leading to our assertion that these miRNAs are master temporal control genes. Another target is the C. elegans homologue of the human proto-oncogene RAS (see below).

We have shown that both the lin-4 and let-7 RNAs are transcriptionally regulated and begin to be expressed at critical times in development, just prior to the down-regulation of their targets. Developmental timing can therefore be distilled down to the timing of expression of these RNAs - we are interested in what controls their transcription. We have dissected the promoter regions of lin-4 and let-7 and have identified a temporal control element in the let-7 promoter that binds a protein that we are pursuing. We are also investigating the role of additional temporally regulated microRNAs during C. elegans development, including 3 let-7 homologues and a lin-4 homologue. We find that members of these families are not co-expressed, suggesting non-redundant functions in various tissues and developmental times.

Mis-regulation of genes that control cell proliferation and cell fate determination often contributes to cancer development. In C. elegans, let-7 controls the timing of proliferation versus differentiation decisions by epidermal cells. In let-7 mutants, cells frequently fail to terminally differentiate, and instead elect to divide again, a hallmark of cancer. In C. elegans, let-7 directly regulates RAS, and another gene, lin-41, which is homologous to cancer genes, including PML, mutated in almost all cases of promylocytic leukemia. let-7 is conserved in humans, where it has been linked to cancer. Specifically, human let-7 is poorly expressed or deleted in lung cancer, and over-expression of let-7 in lung cancer cells inhibits their growth, demonstrating a role for let-7 as a tumor suppressor in lung tissue. We have also shown that human let-7 is expressed in the lung and regulates the expression of important oncogenes implicated in lung cancer, including RAS. We are focusing on the role of let-7 in regulating proto-oncogene expression during lung development and cancer. We are also testing whether over-expression of let-7 suppresses activating oncogenic mutations in RAS, as it does in C. elegans.

Lung cancer is the leading cause of cancer deaths in the US, and the survival rate for patients remains extremely poor. To improve treatment, better understanding of the disease and better therapies are needed. Just as C. elegans pioneered the discoveries that led to the identification of let-7 as a human lung tumor suppressor that regulates RAS, we expect that what we learn about these new genes in C. elegans will provide valuable information on their role in lung cancer. We believe that this work could lead to the identification of novel cancer genes, and shed light on the mechanisms of lung cancer. While this is still an emerging field, the potential benefit to our understanding miRNAs is potentially enormous, not only as cancer loci, but also because of the possibility of using miRNAs as therapeutics in treating lung cancer, e.g. by repressing expression of RAS.

2. the temporal patterning role of the HBL-1 transcription factor and the LIN-41 RING finger protein. By genetic arguments, hbl-1 and lin-41 are major targets of the let-7 RNA, and there are let-7 complementary sequences in the 3'UTR of hbl-1 and lin-41 that are responsive to let-7. While let-7 mutations lead to a reiteration of larval cell fates in the adult animal (in this case cells divide instead of terminally differentiate), hbl-1 and lin-41 mutations display the opposite phenotype and precociously express adult fates in the larval animals (cells terminally differentiate instead of proliferate). hbl-1 and lin-41 encode switches that must be tripped to progress from early fates to later fates. LIN-41 encodes a protein that belongs to a large super family that includes many human oncogenes and tumor suppressor genes and Drosophila Brain Tumor (BRAT). LIN-41 provides temporal cues that allow cells to decide whether to divide or terminally differentiate. We are examining the mechanism of action of LIN-41 and HBL-1. We have performed independent screen for suppressors of both lin-41 and hbl-1 loss of function mutants, and have cloned a number of suppressors genes. We are characterizing these at the moment. We have also followed up a candidate approach, making use of an observation that in Drosophila, the LIN-41 homologue BRAT interacts with PUMILIO to regulate Hunchback, the HBL-1 homologue. In C. elegans the pumilio homologue, puf-9 suppresses lin-41 mutations, but not hbl-1 mutations. We are currently trying to pull together these observations by attempting to show molecularly that lin-41 regulates puf-9 which, in turn regulates hbl-1.

An emerging theme is of universal patterning mechanisms acting throughout the animal kingdom. We are testing this idea by knocking out lin-41 in the mouse. We also find that mlin-41 contains let-7 complementary sites in its 3'UTR and is reciprocally expressed with let-7 during development. We are currently testing whether mlin-41 is regulated by let-7, as it is in C. elegans.

3. the role of the novel, nuclear LIN-14 morphogen in temporal patterning and life-span. The lin-14 gene produces a temporal gradient of LIN-14 protein, such that early in the L1 stage LIN-14 protein levels are high and at the end of the L1 stage LIN-14 protein levels are low. The drop in LIN-14 concentration is controlled by binding of the lin-4 miRNA, to complimentary sites in the lin-14 3'UTR, which trips a switch that allows cells to adopt post-L1 cell fates. Inappropriate expression of LIN-14 at post-L1 times results in a reiteration of L1 fates, while elimination of LIN-14 during the L1 results in L2 fates being expressed precociously. We have little idea how LIN-14 functions to specify the correct timing of cell fate but we have candidate downstream genes and genes whose products interact with LIN-14 to provide us with clues, which we are pursuing.

lin-4 mutants also display striking defects in timing of neural differentiation and display a short lifespan. We have identified targets of lin-4 that control these two developmental defects and are proving the interactions molecularly.

4. A link between developmental timing, circadian timing and cancer. lin-4 and let-7 mutants can be partially suppressed by mutations in the C. elegans homolgue of the Drosophila and mammalian PERIOD homologue, lin-42. This work has led us to investigate an interesting connection between developmental timing and circadian timing. We have shown that many of the components of the circadian clock also control developmental timing in C. elegans. We are now attempting to use these observations to describe a potential mechanism for developmental timing. We would like to link this mechanism to timing of expression of miRNAs to close the circle in the C. elegans work.

Molecular genetic analysis of the heterochronic gene pathway revealed explicit genetic control of temporal patterning in C. elegans that is analogous to the dedicated genetic pathways that control spatial patterning in other metazoans. Moreover, the involvement of antisense microRNAs in translational control during development were the first examples of a more universal mechanism.

Figure 2: Cell lineage pattern defects of the T blast cell associated with lin-14 and lin-4 mutations. The vertical axis represents time. The dashed line represents reiterations of the lineage pattern.

Figure 4: Lineage defects in the lateral hypodermal seam cells associated with let-7 and lin-41 mutants. Unlike in wild-type animals, seam cells in let-7 animals fail to terminally differentiate (represented by 3 horizontal bars) at the beginning of the adult stage, and instead reiterate the larval fate and divide again (the same is true in animals over-expressing lin-41). In contrast, seam cells in lin-41 mutants terminally differentiate one stage earlier than normal (the same is true in animals over-expressing let-7).