Autoregulatory Region Research Articles

Novel Variant Identified in the Enhancer Region of Host Transcription Factor, BRN3A, is a Significant Risk Factor for HPV-Induced Uterine Cervix Cancer.

Among the HPV-mediated cervical cancers, cellular factor BRN3A has gained considerable attention due to its role in promoting an anti-apoptotic cellular environment and in facilitating epitheliotropic transformations of the host. The majority of previous studies looked at BRN3A's molecular characteristics; however, the possibility of genetic variations in BRN3A's auto-regulatory region in relation to cervical cancer risk has been underestimated until now. In a retrospective study in the Eastern UP population, India, we detected genetic variations in the cis-regulatory proximal enhancer region located around 5.6 kb upstream of transcription start site of BRN3A. Our analysis of PCR and DNA sequencing confirmed this novel SNP (BRN3A g.60163379A>G) within the auto-regulatory region of BRN3A. As compared to control subjects, cancer cases exhibited a 1.32-fold higher allele frequency (χ2 = 6.315, p = 0.012). In homozygous (GG) but not in heterozygous conditions, odds ratio (OR) analysis suggests a significant association of cancer risk with the SNP (OR = 2.60, p ≤ 0.004). We further confirmed using the functional analysis that this SNP increased the luciferase gene activity in HPV-positive cervical cancer SiHa cells that were exposed to progesterone. As a result of the association of polymorphisms in a non-coding region of an oncogene with increased cancer risks, we are suggesting that this genetic variation in non-coding region can be used in prediction, diagnosis, or predicting the progression of the disease.

Age-related demethylation of the TDP-43 autoregulatory region in the human motor cortex

In amyotrophic lateral sclerosis (ALS), TAR DNA-binding protein 43 (TDP-43), which is encoded by TARDBP, forms aggregates in the motor cortex. This aggregate formation may be triggered by an increase in the TDP-43 level with aging. However, the amount of TDP-43 is autoregulated by alternative splicing of the TARDBP 3′UTR, and how this autoregulation is affected by aging remains to be elucidated. We found that DNA demethylation in the autoregulatory region in the TARDBP 3′UTR reduced alternative splicing and increased TARDBP mRNA expression. Furthermore, in the human motor cortex, we found that this region was demethylated with aging, resulting in increased expression of TARDBP mRNA. The acceleration of DNA demethylation in the motor cortex was associated with the age of ALS onset. In summary, the dysregulation of TDP-43 autoregulation by age-related DNA demethylation in the motor cortex may explain the contribution of aging and motor system selectivity in ALS.

Actin reorganization at the centrosomal area and the immune synapse regulates polarized secretory traffic of multivesicular bodies in T lymphocytes

ABSTRACT T-cell receptor stimulation induces the convergence of multivesicular bodies towards the microtubule-organizing centre (MTOC) and the polarization of the MTOC to the immune synapse (IS). These events lead to exosome secretion at the IS. We describe here that upon IS formation centrosomal area F-actin decreased concomitantly with MTOC polarization to the IS. PKCδ-interfered T cell clones showed a sustained level of centrosomal area F-actin associated with defective MTOC polarization. We analysed the contribution of two actin cytoskeleton-regulatory proteins, FMNL1 and paxillin, to the regulation of cortical and centrosomal F-actin networks. FMNL1 phosphorylation and F-actin reorganization at the IS were inhibited in PKCδ-interfered clones. F-actin depletion at the central region of the IS, a requirement for MTOC polarization, was associated with FMNL1 phosphorylation at its C-terminal, autoregulatory region. Interfering all FMNL1 isoforms prevented MTOC polarization; nonetheless, FMNL1 re-expression restored MTOC polarization in a centrosomal area F-actin reorganization-independent manner. Moreover, PKCδ-interfered clones exhibited decreased paxillin phosphorylation at the MTOC, which suggests an alternative actin cytoskeleton regulatory pathway. Our results infer that PKCδ regulates MTOC polarization and secretory traffic leading to exosome secretion in a coordinated manner by means of two distinct pathways, one involving FMNL1 regulation and controlling F-actin reorganization at the IS, and the other, comprising paxillin phosphorylation potentially controlling centrosomal area F-actin reorganization. Abbreviations Ab, antibody; AICD, activation-induced cell death; AIP, average intensity projection; APC, antigen-presenting cell; BCR, B-cell receptor for antigen; C, centre of mass; cent2, centrin 2; cIS, central region of the immune synapse; CMAC, CellTracker™ Blue (7-amino-4-chloromethylcoumarin); cSMAC, central supramolecular activation cluster; CTL, cytotoxic T lymphocytes; DAG, diacylglycerol; DGKα, diacylglycerol kinase α; Dia1, Diaphanous-1; dSMAC, distal supramolecular activation cluster; ECL, enhanced chemiluminescence; ESCRT, endosomal sorting complex required for traffic; F-actin, filamentous actin; Fact-low cIS, F-actin-low region at the centre of the immune synapse; FasL, Fas ligand; FMNL1, formin-like 1; fps, frames per second; GFP, green fluorescent protein; HBSS, Hank’s balanced salt solution; HRP, horseradish peroxidase; ILV, intraluminal vesicles; IS, immune synapse; MFI, mean fluorescence intensity; MHC, major histocompatibility complex; MIP, maximal intensity projection; MVB, multivesicular bodies; MTOC, microtubule-organizing centre; NS, not significant; PBL, peripheral blood lymphocytes; PKC, protein kinase C; PKCδ, protein kinase C δ isoform; PLC, phospholipase C; PMA, phorbol myristate acetate; Pol. Index, polarization index; pSMAC, peripheral supramolecular activation cluster; PSF, point spread function; ROI, region of interest; SD, standard deviation; shRNA, short hairpin RNA; SEE, Staphylococcus enterotoxin E; SMAC, supramolecular activation cluster; TCR, T-cell receptor for antigen; T-helper (Th); TRANS, transmittance; WB, Western blot.

Chemical Inhibition of Histone Deacetylases 1 and 2 Induces Fetal Hemoglobin through Activation of GATA2.

Therapeutic intervention aimed at reactivation of fetal hemoglobin protein (HbF) is a promising approach for ameliorating sickle cell disease (SCD) and β-thalassemia. Previous studies showed genetic knockdown of histone deacetylase (HDAC) 1 or 2 is sufficient to induce HbF. Here we show that ACY-957, a selective chemical inhibitor of HDAC1 and 2 (HDAC1/2), elicits a dose and time dependent induction of γ-globin mRNA (HBG) and HbF in cultured primary cells derived from healthy individuals and sickle cell patients. Gene expression profiling of erythroid progenitors treated with ACY-957 identified global changes in gene expression that were significantly enriched in genes previously shown to be affected by HDAC1 or 2 knockdown. These genes included GATA2, which was induced greater than 3-fold. Lentiviral overexpression of GATA2 in primary erythroid progenitors increased HBG, and reduced adult β-globin mRNA (HBB). Furthermore, knockdown of GATA2 attenuated HBG induction by ACY-957. Chromatin immunoprecipitation and sequencing (ChIP-Seq) of primary erythroid progenitors demonstrated that HDAC1 and 2 occupancy was highly correlated throughout the GATA2 locus and that HDAC1/2 inhibition led to elevated histone acetylation at well-known GATA2 autoregulatory regions. The GATA2 protein itself also showed increased binding at these regions in response to ACY-957 treatment. These data show that chemical inhibition of HDAC1/2 induces HBG and suggest that this effect is mediated, at least in part, by histone acetylation-induced activation of the GATA2 gene.

Formin-like 2 Promotes β1-Integrin Trafficking and Invasive Motility Downstream of PKCα

Regulated turnover of integrin receptors is essential for cell adhesion and migration. Pathways selectively regulating β1-integrin recycling are implicated in cancer invasion and metastasis, yet proteins required for the internalization of this pro-invasive integrin remain to be identified. Here, we uncover formin-like 2 (FMNL2) as a critical regulator of β1-integrin internalization downstream of protein kinase C (PKC). PKCα associates with and phosphorylates FMNL2 at S1072 within its Diaphanous autoregulatory region, leading to the release of formin autoinhibition. Phosphorylation of FMNL2 triggers its rapid relocation and promotes its interaction with the cytoplasmic tails of the α-integrin subunits for β1-integrin endocytosis. FMNL2 drives β1-integrin internalization and invasive motility in a phosphorylation-dependent manner, while a FMNL2 mutant defective in actin assembly interferes with β1-integrin endocytosis and cancer cell invasion. Our data establish a role for FMNL2 in the regulation of β1-integrin and provide a mechanistic understanding of the function of FMNL2 in cancer invasiveness.

Acute kidney injury.

Acute kidney injury.

Assessing Cerebral Autoregulation via Oscillatory Lower Body Negative Pressure and Projection Pursuit Regression

The process by which cerebral perfusion is maintained constant over a wide range of systemic pressures is known as “cerebral autoregulation.” Effective dampening of flow against pressure changes occurs over periods as short as ~15 sec and becomes progressively greater over longer time periods. Thus, slower changes in blood pressure are effectively blunted and faster changes or fluctuations pass through to cerebral blood flow relatively unaffected. The primary difficulty in characterizing the frequency dependence of cerebral autoregulation is the lack of prominent spontaneous fluctuations in arterial pressure around the frequencies of interest (less than ~0.07 Hz or ~15 sec). Oscillatory lower body negative pressure (OLBNP) can be employed to generate oscillations in central venous return that result in arterial pressure fluctuations at the frequency of OLBNP. Moreover, Projection Pursuit Regression (PPR) provides a nonparametric method to characterize nonlinear relations inherent in the system without a priori assumptions and reveals the characteristic non-linearity of cerebral autoregulation. OLBNP generates larger fluctuations in arterial pressure as the frequency of negative pressure oscillations become slower; however, fluctuations in cerebral blood flow become progressively lesser. Hence, the PPR shows an increasingly more prominent autoregulatory region at OLBNP frequencies of 0.05 Hz and below (20 sec cycles). The goal of this approach it to allow laboratory-based determination of the characteristic nonlinear relationship between pressure and cerebral flow and could provide unique insight to integrated cerebrovascular control as well as to physiological alterations underlying impaired cerebral autoregulation (e.g., after traumatic brain injury, stroke, etc.).

Assessing cerebral autoregulation via oscillatory lower body negative pressure and projection pursuit regression.

The process by which cerebral perfusion is maintained constant over a wide range of systemic pressures is known as “cerebral autoregulation.” Effective dampening of flow against pressure changes occurs over periods as short as ~15 sec and becomes progressively greater over longer time periods. Thus, slower changes in blood pressure are effectively blunted and faster changes or fluctuations pass through to cerebral blood flow relatively unaffected. The primary difficulty in characterizing the frequency dependence of cerebral autoregulation is the lack of prominent spontaneous fluctuations in arterial pressure around the frequencies of interest (less than ~0.07 Hz or ~15 sec). Oscillatory lower body negative pressure (OLBNP) can be employed to generate oscillations in central venous return that result in arterial pressure fluctuations at the frequency of OLBNP. Moreover, Projection Pursuit Regression (PPR) provides a nonparametric method to characterize nonlinear relations inherent in the system without a priori assumptions and reveals the characteristic non-linearity of cerebral autoregulation. OLBNP generates larger fluctuations in arterial pressure as the frequency of negative pressure oscillations become slower; however, fluctuations in cerebral blood flow become progressively lesser. Hence, the PPR shows an increasingly more prominent autoregulatory region at OLBNP frequencies of 0.05 Hz and below (20 sec cycles). The goal of this approach it to allow laboratory-based determination of the characteristic nonlinear relationship between pressure and cerebral flow and could provide unique insight to integrated cerebrovascular control as well as to physiological alterations underlying impaired cerebral autoregulation (e.g., after traumatic brain injury, stroke, etc.).

Relative contributions of sympathetic, cholinergic, and myogenic mechanisms to cerebral autoregulation.

Prior work aimed at improving our understanding of human cerebral autoregulation has explored individual physiological mechanisms of autoregulation in isolation, but none has attempted to consolidate the individual roles of these mechanisms into a comprehensive model of the overall cerebral pressure-flow relationship. We retrospectively analyzed this relationship before and after pharmacological blockade of α-adrenergic-, muscarinic-, and calcium channel-mediated mechanisms in 43 healthy volunteers to determine the relative contributions of the sympathetic, cholinergic, and myogenic controllers to cerebral autoregulation. Projection pursuit regression was used to assess the effect of pharmacological blockade on the cerebral pressure-flow relationship. Subsequently, ANCOVA decomposition was used to determine the cumulative effect of these 3 mechanisms on cerebral autoregulation and whether they can fully explain it. Sympathetic, cholinergic, and myogenic mechanisms together accounted for 62% of the cerebral pressure-flow relationship (P<0.05), with significant and distinct contributions from each of the 3 effectors. ANCOVA decomposition demonstrated that myogenic effectors were the largest determinant of the cerebral pressure-flow relationship, but their effect was outside of the autoregulatory region where neurogenic control appeared prepotent. Our results suggest that myogenic effects occur outside the active region of autoregulation, whereas neurogenic influences are largely responsible for cerebral blood flow control within it. However, our model of cerebral autoregulation left 38% of the cerebral pressure-flow relationship unexplained, suggesting that there are other physiological mechanisms that contribute to cerebral autoregulation.

The cerebovasculature: a smooth (muscle) operator?

The cerebovasculature: a smooth (muscle) operator?

Neurohormonal interactions on the renal oxygen delivery and consumption in haemorrhagic shock-induced acute kidney injury

Haemorrhagic shock is a common cause of acute kidney injury (AKI), which is a major risk factor for developing chronic kidney disease. The mechanism is superficially straightforward. An arterial pressure below the kidney's autoregulatory region leads to a direct reduction in filtration pressure and perfusion, which in turn cause renal failure with reduced glomerular filtration rate and AKI because of hypoxia. However, the kidney's situation is further worsened by the hormonal and neural reactions to reduced perfusion pressure. There are three major systems working to maintain arterial pressure in shock: sympathetic signalling, the renin-angiotensin system and vasopressin. These work to retain electrolytes and water and to increase peripheral resistance and cardiac output. In the kidney, the increased electrolyte reabsorption consumes oxygen. At the same time, at the signalling level seen in shock, all of these hormones reduce renal perfusion and thereby oxygen delivery. This creates an exaggerated hypoxic situation that is liable to worsen the AKI. The present review will examine this mechanistic background and identify a number of areas that require further studies. At this time, the ideal treatment of haemorrhagic shock appears to be slow fluid resuscitation, possibly with hyperosmolar sodium, low chloride and no artificial colloids. From the standpoint of the kidney, renin-angiotensin system inhibitors appear fruitful for further study.

Defining the physiology of cerebral autoregulation

Cerebrovascular autoregulation is critical for neurophysiologic health; however, our understanding of the mechanisms is limited. Recent studies show the importance of noradrenergic (NA) and cholinergic (ACh) pathways, though less so for myogenic (MYO). There have been no attempts to consolidate these effectors of cerebral flow into a comprehensive model. Moreover, most work relies on linear approaches that are limited when applied to this inherently nonlinear function. Therefore, we used our projection pursuit method to explore alterations in the relation between pressure (Finapres) and cerebral flow (Doppler) in unblocked (n=48) and blocked (NA, n=11; ACh, n=9; MYO, n=16) states. Differences in autoregulation caused by blockade were used to derive a predictive model for autoregulation in the unblocked state. Projection pursuit provided gain and range of autoregulation as well as the pressure‐flow relations above and below this range. NA blockade markedly linearized the autoregulatory response whereas ACh blockade primarily increased the autoregulatory range by 50%. In contrast to what has been found with linear assessments, MYO blockade reduced the pressure‐flow relations above and below the autoregulatory region. Thus, the predictive model used the contribution of each individual mechanism to achieve an integrated understanding of cerebral autoregulatory control. Support: NIH HL093113.

Defining the characteristic relationship between arterial pressure and cerebral flow

Reliable assessment of cerebrovascular effectiveness in buffering against pressure fluctuations may have important implications for the timing and the outcome of therapy after adverse cerebrovascular events. Although linear approaches may indicate the presence or absence of cerebral autoregulation, they are inadequate to describe its characteristics and its effectiveness. Establishing a simple yet robust methodology to reliably measure the effectiveness of cerebral autoregulation could provide a tool to guide screening and clinical options to characterize and treat adverse cerebrovascular events associated with alterations in cerebral perfusion. To test the utility of one such methodology, an oscillatory lower body negative pressure of 30-40 mmHg was used at six frequencies from 0.03 to 0.08 Hz in 43 healthy volunteers, and the pressure-flow relation and the effectiveness of autoregulation was quantified using projection pursuit regression. Projection pursuit regression explained the majority of the relationship between pressure and cerebral blood flow fluctuations and revealed its nature consistently across individuals and across separate study days. The nature of this relationship entailed an autoregulatory region wherein slow arterial pressure fluctuations are effectively counterregulated and two passive regions wherein pressure fluctuations resulted in parallel changes in flow. The effectiveness of autoregulation was significantly reduced as pressure fluctuations became faster. These results demonstrate the characteristic relationship between arterial pressure and cerebral blood flow. Furthermore, the methodology utilized in this study provides a tool that can provide unique insight to integrated cerebrovascular control and may allow diagnosis of physiological alterations underlying impaired cerebral autoregulation.

CaMKII Autonomy Is Substrate-dependent and Further Stimulated by Ca2+/Calmodulin

A hallmark feature of Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) regulation is the generation of Ca(2+)-independent autonomous activity by Thr-286 autophosphorylation. CaMKII autonomy has been regarded a form of molecular memory and is indeed important in neuronal plasticity and learning/memory. Thr-286-phosphorylated CaMKII is thought to be essentially fully active ( approximately 70-100%), implicating that it is no longer regulated and that its dramatically increased Ca(2+)/CaM affinity is of minor functional importance. However, this study shows that autonomy greater than 15-25% was the exception, not the rule, and required a special mechanism (T-site binding; by the T-substrates AC2 or NR2B). Autonomous activity toward regular R-substrates (including tyrosine hydroxylase and GluR1) was significantly further stimulated by Ca(2+)/CaM, both in vitro and within cells. Altered K(m) and V(max) made autonomy also substrate- (and ATP) concentration-dependent, but only over a narrow range, with remarkable stability at physiological concentrations. Such regulation still allows molecular memory of previous Ca(2+) signals, but prevents complete uncoupling from subsequent cellular stimulation.

EWS/ETS proteins promote expression and regulate function of the homeodomain transcription factor BRN3A

Ewing's sarcoma family tumors (ESFTs or EFTs) express neuronal markers, which indicates they may originate from cells at least partly committed to neuronal lineage. However, recent publications suggest EFT originates in mesenchymal stem cells, and EWS/ETS fusion proteins characteristic of EFT activate neuronal marker expression to confer a neural phenotype on EFT. Here we show that the neuronal marker BRN3A/POU4F1 is expressed abundantly at the protein level in primary EFT but not in rhabdomyosarcoma and neuroblastoma, and EFT cells exhibit high activity of the BRN3A proximal autoregulatory region. EWS/FLI-1 siRNA reduces BRN3A expression and promoter activity and EWS/ETS proteins are bound to the BRN3A locus, suggesting a direct function for EWS/ETS proteins in control of BRN3A expression. Differentiation-associated and autoregulatory activities of BRN3A are respectively impaired and altered in EFT cells, and EWS/FLI-1 siRNA can restore some BRN3A function. A potentially novel function for BRN3A in EFT cells is identified. These results extend the hypothesis that EWS/ETS proteins induce expression of neuronal markers such as BRN3A in EFT by showing that the function of those same markers may be restricted or controlled in an EWS/ETS-dependent manner.

Phosphatidylinositol-4,5-Bisphosphate and Phospholipase D-Generated Phosphatidic Acid Specify SNARE-Mediated Vesicle Fusion for Prospore Membrane Formation

The soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) family of proteins is required for eukaryotic intracellular membrane fusions. Vesicle fusion for formation of the prospore membrane (PSM), a membrane compartment that forms de novo during yeast sporulation, requires SNARE function, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P(2)], and the activity of the phospholipase D (PLD) Spo14p, which generates phosphatidic acid (PA). The SNARE syntaxin Sso1p is essential for PSM production while the functionally redundant homolog in vegetative growth, Sso2p, is not. We demonstrate that Sso1p and Sso2p bind similarly in vitro to PA or phosphoinositide-containing liposomes and that the conserved SNARE (H3) domain largely mediates PA-binding. Both green fluorescent protein-Sso fusion proteins localize to the developing PSM in wild-type cells and to the spindle pole body in spo14Delta cells induced to sporulate. However, the autoregulatory region of Sso1p binds PI(4,5)P(2)-containing liposomes in vitro with a greater ability than the equivalent region of Sso2p. Overexpression of the phosphatidylinositol-4-phosphate 5-kinase MSS4 in sso1Delta cells induced to sporulate stimulates PSM production; PLD activity is not increased under these conditions, indicating that PI(4,5)P(2) has roles in addition to stimulating PLD in PSM formation. These data suggest that PLD-generated PA and PI(4,5)P(2) collaborate at multiple levels to promote SNARE-mediated fusion for PSM formation.

Comparative analysis of CAX2-like cation transporters indicates functional and regulatory diversity

Internal compartmentalization of metals is an important metal tolerance mechanism in many organisms. In plants and fungi, sequestration into the vacuole is a major detoxification mechanism for metals. Cation transport into the vacuole can be mediated by CAX (cation exchanger) transporters. The Arabidopsis thaliana AtCAX2 transporter was shown previously to transport Ca(2+), Cd(2+) and Mn(2+). To assess the conservation of the functional and regulatory characteristics of CAX2-like transporters in higher plants, we have characterized AtCAX2 orthologues from Arabidopsis (AtCAX5), tomato (LeCAX2) and barley (HvCAX2). Substrate specificity and regulatory activity were assessed using a yeast heterologous-expression assay. Each CAX could transport Ca(2+) and Mn(2+) into the yeast vacuole, but they each had different cation transport kinetics. Most notably, there was variation in the regulation of the transporters. As found with AtCAX2 previously, only expression of an N-terminally truncated form of AtCAX5 in yeast was able to mediate Ca(2+) and Mn(2+) transport, indicating that activity may be controlled by an autoregulatory region at the N-terminus. In contrast, either full-length or truncated LeCAX2 could efficiently transport Ca(2+), although Mn(2+) transport was controlled by the N-terminus. HvCAX2 did not appear to possess an N-terminal regulatory domain. Expression of AtCAX2 was not significantly modulated by metal stress; however, AtCAX5 and HvCAX2 were transcriptionally up-regulated by high Mn(2+) treatment, and by Ca(2+) and Na(+) stress respectively. It is therefore apparent that, despite the high sequence identity between plant CAX2 orthologues, there is significant diversity in their functional characteristics, particularly with regard to regulatory mechanisms.

Structure of a BCOR Corepressor Peptide in Complex with the BCL6 BTB Domain Dimer

The transcriptional corepressors BCOR, SMRT, and NCoR are known to bind competitively to the BCL6 BTB domain despite the fact that BCOR has no detectable sequence similarity to the other two corepressors. We have identified a 17 residue motif from BCOR that binds directly to the BCL6 BTB domain and determined the crystal structure of the complex to a resolution of 2.6 A. Remarkably, the BCOR BCL6 binding domain (BCOR(BBD)) peptide binds in the same BCL6 binding site as the SMRT(BBD) peptide despite the lack of any significant sequence similarity between the two peptides. Mutations of critical BCOR(BBD) residues cause the disruption of the BCL6 corepression activities of BCOR, and a BCOR(BBD) peptide blocks BCL6-mediated transcriptional repression and kills lymphoma cells.

A Rac-cGMP Signaling Pathway

The small GTPase Rac and the second messenger cGMP (guanosine 3',5'-cyclic monophosphate) are critical regulators of diverse cell functions. When activated by extracellular signals via membrane signaling receptors, Rac executes its functions through engaging downstream effectors such as p21-activated kinase (PAK), a serine/threonine protein kinase. However, the molecular mechanism by which membrane signaling receptors regulate cGMP levels is not known. Here we have uncovered a signaling pathway linking Rac to the increase of cellular cGMP. We show that Rac uses PAK to directly activate transmembrane guanylyl cyclases (GCs), leading to increased cellular cGMP levels. This Rac/PAK/GC/cGMP pathway is involved in platelet-derived growth factor-induced fibroblast cell migration and lamellipodium formation. Our findings connect two important regulators of cellular physiological functions and provide a general mechanism for diverse receptors to modulate physiological responses through elevating cellular cGMP levels.

Identification of specific protein regions involved in the regulation of the Diaphanous‐related Formin, DAAM1

Diaphanous-related formins (DRF) are a conserved family of proteins that are involved in the coordination of the cytoskeleton. Based on studies of yeast and mammalian DRF proteins, the regulation of DRFs are dependent on the mechanism of autoinhibition; the Dia-autoregulatory domain (DAD) binds to the Diaphanous inhibitory domain (DID), keeping the DRF inactive until Rho GTPases binds to the GTPase Binding Domain (GBD) and releases the DAD from DID, thereby activating the DRF protein. Since DID-DAD binding plays an important role in dictating the regulation of DRFs, our laboratory has focused on elucidating the autoregulatory regions in another DRF family member, DAAM1 (Dishevelled-associated activator of morphogenesis). Known to interact with the GTPases RhoA and Cdc42, DAAM1 has also been shown to work with Src in the formation of branched protrusions of the cell. Here, we have used a combination of somatic cell microinjection, immunofluorescence, and fluorescence anisotropy to elucidate the specific regions involved in the autoregulation of DAAM1. In addition, we have identified a specific residue in the DAD region that may play a pivotal role in DAAM1 autoregulation.