Cholesterol homeostasis: clipping out a slippery regulator.

SREBPs are transcriptional activators central to cholesterol homeostasis. Recent work has shown that a two-step cleavage of membrane-bound SREBPs frees them to enter the nucleus. An activator of the first, sterol-regulated proteolysis step has also been identified.

Promoter selective transcriptional synergy mediated by sterol regulatory element binding protein and Sp1: a critical role for the Btd domain of Sp1.

Cellular cholesterol and fatty acid levels are coordinately regulated by a family of transcriptional regulatory proteins designated sterol regulatory element binding proteins (SREBPs). SREBP-dependent transcriptional activation from all promoters examined thus far is dependent on the presence of an additional binding site for a ubiquitous coactivator. In the low-density lipoprotein (LDL) receptor, acetyl coenzyme A carboxylase (ACC), and fatty acid synthase (FAS) promoters, which are all regulated by SREBP, the coactivator is the transcription factor Sp1. In this report, we demonstrate that Sp3, another member of the Sp1 family, is capable of substituting for Sp1 in coactivating transcription from all three of these promoters. Results of an earlier study showed that efficient activation of transcription from the LDL receptor promoter required domain C of Sp1; however, this domain is not crucial for activation of the simian virus 40 promoter, where synergistic activation occurs through multiple Sp1 binding sites and does not require SREBP. Also in the present report, we further localize the critical determinant of the C domain required for activation of the LDL receptor to a small region that is highly conserved between Sp1 and Sp3. This crucial domain encompasses the buttonhead box, which is a 10-amino-acid stretch that is present in several Sp1 family members, including the Drosophila buttonhead gene product. Interestingly, neither the buttonhead box nor the entire C domain is required for the activation of the FAS and ACC promoters even though both SREBP and Sp1 are critical players. ACC and FAS each contain two critical SREBP sites, whereas there is only one in the LDL receptor promoter. This finding suggested that buttonhead-dependent activation by SREBP and Sp1 may be limited to promoters that naturally contain a single SREBP recognition site. Consistent with this model, a synthetic construct containing three tandem copies of the native LDL receptor SREBP site linked to a single Sp1 site was also significantly activated in a buttonhead-independent fashion. Taken together, these studies indicate that transcriptional activation through the concerted action of SREBP and Sp1 can occur by at least two different mechanisms, and promoters that are activated by each one can potentially be identified by the number of critical SREBP binding sites that they contain.

Transcription control region within the protein-coding portion of adenovirus E1A genes.

A single-base deletion within the protein-coding region of the adenovirus type 5 early region 1A (E1A) genes, 399 bases downstream from the transcription start site, depresses transcription to 2% of the wild-type rate. Complementation studies demonstrated that this was due to two effects of the mutation: first, inactivation of an E1A protein, causing a reduction by a factor of 5; second, a defect which acts in cis to depress E1A mRNA and nuclear RNA concentrations by a factor of 10. A larger deletion within the protein-coding region of E1A which overlaps the single-base deletion produces the same phenotype. In contrast, a linker insertion which results in a similar truncated E1A protein does not produce the cis-acting defect in E1A transcription. These results demonstrate that a critical cis-acting transcription control region occurs within the protein coding sequence in adenovirus type 5 E1A. The single-base deletion occurs in a sequence which shows extensive homology with a sequence from the enhancer regions of simian virus 40 and polyomavirus. This region is not required for E1A transcription during the late phase of infection.

Conditional response of the human steroidogenic acute regulatory protein gene promoter to sterol regulatory element binding protein-1a.

The steroidogenic acute regulatory protein (StAR) gene controls the rate-limiting step in the biogenesis of steroid hormones, delivery of cholesterol to the cholesterol side-chain cleavage enzyme on the inner mitochondrial membrane. We determined whether the human StAR promoter is responsive to sterol regulatory element-binding proteins (SREBPs). Expression of SREBP-1a stimulated StAR promoter activity in the context of COS-1 cells and human granulosa-lutein cells. In contrast, expression of SREBP-2 produced only a modest stimulation of StAR promoter activity. One of the SREBP-1a response elements in the StAR promoter was mapped in deletion constructs and by site-directed mutagenesis between nucleotides -81 to -70 from the transcription start site. This motif bound recombinant SREBPs in electrophoretic mobility shift assays, but with lesser affinity than a low density lipoprotein receptor SREBP-binding site. An additional binding site for the transcriptional modulator, yin yang 1 (YY1), was observed within the SREBP-binding site (nucleotides -73 to -70). Mutation of the YY1-binding site increased the responsiveness of the StAR promoter to exogenous SREBP-1a, but did not alter the affinity for SREBP-1a binding in electrophoretic mobility gel shift assays. Manipulations that altered endogenous mature SREBP-1a levels (e.g. culture in lipoprotein-deficient medium and addition of 27-hydroxycholesterol) did not affect StAR promoter function, but influenced low density lipoprotein receptor promoter activity. We conclude that 1) the human StAR promoter is conditionally responsive to SREBP-1a such that promoter activity is up-regulated in the presence of high levels of SREBP-1a, but is unaffected when mature SREBP levels are suppressed; and 2) the human StAR promoter is selectively responsive to SREBP-1a.

Transcriptional control mechanisms in the regulation of cholesterol balance.

The mechanisms that govern regulation of cholesterol metabolism in higher eukaryotic cells provide an example of how metabolic regulation has evolved to establish growth and nutritional control in a multicellular environment. Two sources of cholesterol must be balanced to ensure optimum growth and viability. Much of the control is established by regulating the levels of key proteins involved in cholesterol uptake and biosynthesis and this occurs by alterations in promoter activity. The studies discussed here track the progression in understanding the mechanism for transcriptional regulation by cholesterol from the isolation of the key genes involved, to the careful dissection of the cis-acting sequences that control expression, and on to what is currently known about the trans-acting proteins that mediate the regulatory response.

Single nucleotide resolution of sterol regulatory region in promoter for 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Sterol-dependent regulation of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase promoter was previously localized to a 42-base pair region containing an octamer sequence, referred to as the sterol regulatory element (SRE-1). A similar motif is found in the region of DNA that is required for sterol-dependent regulation of the HMG-CoA synthase and low density lipoprotein receptor genes. Single nucleotide substitution analyses of the low density lipoprotein receptor and HMG-CoA synthase promoters confirmed that the SRE-1 is an important sterol regulatory motif. In the current studies, a series of single nucleotide mutations were introduced into the HMG-CoA reductase regulatory region and transfected into Chinese hamster ovary cells. RNA produced by each mutant promoter was then measured in the presence or absence of sterols. Thirty-seven independent mutations were analyzed, and two separate domains were identified as being critical. One essential region was spread over 10 bases and contained half of the SRE-1; however, the other half of the SRE-1 was not important for sterol regulation. The second essential region spanned four contiguous bases. These two critical elements are separated from each other by three nonessential bases. The results are interpreted to suggest that regulation of HMG-CoA reductase gene transcription by sterols requires additional or possibly separate factors from those required for sterol regulation of the low density lipoprotein receptor and HMG-CoA synthase promoters.

Two separate sites contribute to AP-1 activation of the promoter for 3-hydroxy-3-methylglutaryl coenzyme A synthase.

Metabolic flux into the mevalonate pathway is regulated by end product repression and cell growth. In the experiments reported here the transcriptional promoter for an early enzyme of the pathway, 3-hydroxy-3-methylglutaryl coenzyme A synthase, is shown to be activated by the growth stimulatory agent tetraphorbol acetate (TPA). We show that TPA has a direct stimulatory action on the promoter and further that this is mediated by the AP-1 transcription factor. In addition, we show that there are two separate cis-acting sites that bind AP-1 and both are required for maximal stimulation. We further show that in AP-1-deficient cells ectopic expression of AP-1 stimulates synthetic promoters containing two copies of each synthase element upstream of a minimal promoter. The physiological rationale of having both end product repression and direct activation by growth stimulatory cues is discussed.

Nutrient regulation of gene expression by the sterol regulatory element binding proteins: increased recruitment of gene-specific coregulatory factors and selective hyperacetylation of histone H3 in vivo.

We have evaluated the mechanism for sterol-regulated gene expression by the sterol regulatory element binding proteins (SREBPs) in intact cells. We show that activation of SREBPs by sterol depletion results in the increased binding of Sp1 to a site adjacent to SREBP in the promoter for the low density lipoprotein (LDL) receptor gene in vivo. Similarly, sterol depletion resulted in the increased recruitment of two distinct SREBP coregulatory factors, NF-Y and CREB, to the promoter for hydroxymethyl glutaryl CoA reductase, another key gene of intracellular cholesterol homeostasis. Furthermore, increased acetylation of histone H3 but not H4 was also detected in chromatin from both promoters on SREBP activation. Thus, SREBP activation results in the similar selective recruitment of different coregulatory generic transcription factors to two separate cholesterol-regulated promoters. These studies demonstrate the utility of the chromatin immunoprecipitation technique for analyzing the differential action of low-abundance transcription factors in fundamental regulatory events in intact cells. Our results also provide key in vivo support for the mechanism proposed from cell-free experiments, where SREBP increased the binding of Sp1 to the LDL receptor promoter. Finally, our findings also indicate that subtle differences in the pattern of core histone acetylation play a role in selective gene activation.

Two tandem binding sites for sterol regulatory element binding proteins are required for sterol regulation of fatty-acid synthase promoter.

We previously reported that sterol regulation of the rat fatty-acid synthase was lost when the DNA sequence between -73 and -43 of the promoter was deleted from a luciferase reporter construct (Bennett, M. K., Lopez, J. M., Sanchez, H. B., and Osborne, T. F. (1995) J. Biol. Chem. 270, 25578-25583). We also showed that there was a binding site for sterol regulatory element binding protein-1 (SREBP-1) in this region that contains a palindromic E-box motif (5'-CANNTG-3'). This is the consensus recognition element for basic-helix-loop-helix leucine zipper containing proteins such as the SREBPs. However, the SREBPs are unique basic-helix-loop-helix leucine zipper proteins that not only bind to a subset of E-boxes but also to the direct repeat SRE-1 element of the low density lipoprotein receptor promoter as well as to variant sites present in the promoters for key enzymes of both cholesterol and fatty acid biosynthesis. Based on the sequence of the variant SREBP recognition sites in these other promoters, we noted there was more than one potential recognition site for SREBP within the -73 to -43 interval of the fatty-acid synthase promoter. In the present studies we have systematically mutated these potential SREBP sites and have analyzed the consequences on sterol regulation, activation by exogenously supplied SREBPs, and binding by SREBPs in vitro. The results clearly show that the E-box element is not the SREBP recognition site in this region. Rather, there are two independent SREBP binding sites that flank the E-box, and both are required for maximal sterol regulation and activation by transfected SREBP protein.

Molecular aspects in feedback regulation of gene expression by cholesterol in mammalian cells.

Intracellular cholesterol balance is maintained by a tight feedback mechanism that prevents the overaccumulation of cholesterol to cytotoxic levels. This is achieved through the coordinate regulation of genes of cholesterol uptake and biosynthesis by the sterol regulatory element binding proteins (SREBPs). The SREBPs are synthesized as membrane bound precursors that are released from their membrane tether when the cell needs new cholesterol. In the present article we present a model for how the cholesterol uptake pathway may be activated before the biosynthetic pathway to prevent wasting cellular energy and carbon on unneeded synthesis. Then we introduce a system for analyzing the differential localization and cellular trafficking of the different SREBP isoforms that can be performed over time in living cells.

Far upstream initiation sites for adenovirus early region 1A transcription are utilized after the onset of viral DNA replication.

Adenovirus early region 1A (E1A) is the first transcription unit expressed after infection. It encodes a protein which controls the expression of all other early viral genes. The E1A mRNAs have one major capped 5' terminus which maps 31 nucleotides downstream from a T-A-T-A sequence (C. Baker and E. Ziff. J. Mol. Biol. 149:189-221, 1981). In addition, a minor set of E1A mRNAs are observed during the early phase of infection which have 5' termini mapping at approximately -160, -185, and -230 relative to the major cap site (Osborne et al., Cell 29:139-148, 1982). Here we report the occurrence of another set of minor E1A mRNAs which were observed exclusively after the initiation of viral DNA replication. These late specific E1A mRNAs had cap sites which mapped at approximately -300, -325, -360, and -375 relative to the major cap site. The appearance of these minor late E1A mRNAs was blocked by the DNA synthesis inhibitor cytosine arabinoside. These same late specific E1A mRNAs were synthesized from E1A-containing plasmids which replicate in monkey cells. This demonstrated that neither late specific adenovirus proteins nor adenovirus-specific chromatin structure was required for the production of the late specific E1A mRNAs. Adenovirus mutants in which the E1A T-A-T-A box region had been deleted also synthesized the corresponding deleted forms of the late specific mRNAs after initiation of DNA replication. These results indicate that the process of DNA replication alters the specificity of E1A transcription initiation in a promoter region which is at least 375 nucleotides in length.

HLH106, a Drosophila sterol regulatory element-binding protein in a natural cholesterol auxotroph.

In mammalian cells, sterol regulatory element-binding proteins (SREBPs) coordinate metabolic flux through the cholesterol and fatty acid biosynthetic pathways in response to intracellular cholesterol levels. We describe experiments that evaluate the functional equivalence of mammalian SREBPs and the insect homologue of SREBP-1a, HLH106, in both mammalian and insect cell culture systems. HLH106 binds to both palindromic E-boxes and direct repeat sterol regulatory elements (SREs) efficiently, suggesting that it has a dual DNA binding specificity similar to the mammalian proteins. The amino-terminal "mature" protein activates transcription from mammalian SREs in both mammalian and Drosophila tissue culture cells. Additionally, HLH106 also requires a ubiquitous regulatory co-activator to efficiently activate transcription from mammalian SREs. These properties are shared with its mammalian counterparts. When expressed in mammalian cells, the carboxyl-terminal portion also localizes to perinuclear membranes similar to mammalian SREBPs. Furthermore, membrane-bound HLH106 is proteolytically processed in response to intracellular sterol levels in mammalian cells in an SREBP cleavage-activating protein-stimulated fashion. The presence of an SREBP homologue in Drosophila whose processing is regulated by intracellular sterol levels when expressed in mammalian cells suggests that related processing machinery exists in insect cells. This is notable, since insects are reportedly incapable of de novo sterol biosynthesis.

Related membrane domains in proteins of sterol sensing and cell signaling provide a glimpse of treasures still buried within the dynamic realm of intracellular metabolic regulation.

Recent discoveries in the regulation of cholesterol metabolism have documented a two step proteolytic pathway that regulates nuclear targeting of the sterol regulatory element binding proteins. Sterol regulatory element binding protein cleavage activating protein is a newly identified protein that modulates the proteolytic maturation of the sterol regulatory element binding proteins. It contains a domain that is quite similar in sequence to the membrane spanning region of the rate controlling enzyme of cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase. The membrane domain of the reductase is involved in its post-translational regulation by cholesterol. The molecular defect in the intracellular cholesterol storage disease, Niemann-Pick type C, has also recently been identified. Surprisingly, the affected gene encodes a protein with similarity to the membrane domains that are conserved in 3-hydroxy-3-methylglutaryl reductase and sterol regulatory element binding protein cleavage activating protein. Furthermore, the cell surface receptor for the sterol modified hedgehog morphogen, Patched, also contains a membrane domain with significant similarity to this putative sterol monitoring domain. These recent developments suggest a common mechanism for sensing intracellular sterol levels and cell signaling, which is based on the function of related membrane domains that are contained in key regulatory proteins.

Specificity in cholesterol regulation of gene expression by coevolution of sterol regulatory DNA element and its binding protein.

When demand for cholesterol rises in mammalian cells, the sterol regulatory element (SRE) binding proteins (SREBPs) are released from their membrane anchor through proteolysis. Then, the N-terminal region enters the nucleus and activates genes of cholesterol uptake and biosynthesis. Basic helix-loop-helix (bHLH) proteins such as SREBPs bind to a palindromic DNA sequence called the E-box (5'-CANNTG-3'). However, SREBPs are special because they also bind direct repeat elements called SREs. Importantly, sterol regulation of all promoters studied thus far is mediated by SREBP binding only to SREs. To study the reason for this we converted the direct repeat SRE from the sterol-regulated low-density lipoprotein receptor promoter into an E-box. In this report we show that SREBPs are still able to bind and activate this promoter however, sterol regulation is lost. The results are consistent with the mutant promoter being a target for promiscuous activation by constitutively expressed E-box binding bHLH proteins that are not regulated by cholesterol. Kim and coworkers [Kim, J. B., Spotts, G. D., Halvorsen, Y.-D., Shih, H.-M., Ellenberger, T., Towle, H. C. & Spiegelman, B. M. (1995) Mol. Cell. Biol. 15, 2582-2588] demonstrated that the dual DNA binding specificity of SREBPs is caused by a specific tyrosine in the conserved basic region of the DNA binding domain that corresponds to an arginine in all other bHLH proteins that recognize only E-boxes. Taken together the data suggest an evolutionary mechanism where a DNA binding protein along with its recognition site have coevolved to ensure maximal specificity and sensitivity in a crucial nutritional regulatory response.

Red 25, a protein that binds specifically to the sterol regulatory region in the promoter for 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

A protein that binds to the sterol regulatory region of the hamster promoter for 3-hydroxy-3-methylglutaryl-coenzyme A reductase has been identified. All of the DNA bases crucial to the binding of this protein were previously shown to be essential for sterol regulation of the intact promoter in cultured cells. This low abundance protein, called Red 25, has been purified from nuclear extracts of hamster liver by a series of standard chromatographic techniques coupled with a DNA affinity step. Its size has been estimated as approximately 42 kDa by gel electrophoresis, size exclusion chromatography, and protein-DNA cross-linking studies. Furthermore, it binds to its target site with a Kd = 6 x 10(-11) M. Red 25 does not bind to the sterol regulatory regions of the LDL receptor or 3-hydroxy-3-methylglutaryl-coenzyme A synthase. This is consistent with recent studies that show there is a unique site for sterol regulation in the reductase promoter. The identification and purification of this protein represents a significant step in the study of feedback regulation by cholesterol.

Domains of transcription factor Sp1 required for synergistic activation with sterol regulatory element binding protein 1 of low density lipoprotein receptor promoter.

Feedback regulation of transcription from the low density lipoprotein (LDL) receptor gene is fundamentally important in the maintenance of intracellular sterol balance. The region of the LDL receptor promoter responsible for normal sterol regulation contains adjacent binding sites for the ubiquitous transcription factor Sp1 and the cholesterol-sensitive sterol regulatory element-binding proteins (SREBPs). Interestingly, both are essential for normal sterolmediated regulation of the promoter. The cooperation by Sp1 and SREBP-1 occurs at two steps in the activation process. SREBP-1 stimulates the binding of Sp1 to its adjacent recognition site in the promoter followed by enhanced stimulation of transcription after both proteins are bound to DNA. In the present report, we have defined the protein domains of Sp1 that are required for both synergistic DNA binding and transcriptional activation. The major activation domains of Sp1 that have previously been shown to be essential to activation of promoters containing multiple Sp1 sites are required for activation of the LDL receptor promoter. Additionally, the C domain is also crucial. This slightly acidic approximately 120-amino acid region is not required for efficient synergistic activation by multiple Sp1 sites or in combination with other recently characterized transcriptional regulators. We also show that Sp1 domain C is essential for full, enhanced DNA binding by SREBP-1. Taken together with other recent studies on the role of Sp1 in promoter activation, the current experiments suggest a unique combinatorial mechanism for promoter activation by two distinct transcription factors that are both essential to intracellular cholesterol homeostasis.

Cooperation by sterol regulatory element-binding protein and Sp1 in sterol regulation of low density lipoprotein receptor gene.

Regulation of the low density lipoprotein (LDL) receptor promoter by cholesterol requires a well defined sterol regulatory site and an adjacent binding site for the universal transcription factor Sp1. These elements are located in repeats 2 and 3 of the wild type promoter, respectively. The experiments reported here demonstrate that Sp1 participates in sterol regulation of the LDL receptor in an orientation-specific fashion. We present data which suggest that sterol regulatory element-binding protein (SREBP) increases the binding of Sp1 to the adjacent repeat 3 sequence. We also demonstrate that SREBP and Sp1 synergistically activate expression from the LDL receptor promoter inside the cell by cotransfecting expression vectors encoding each protein into Drosophila tissue culture cells that are devoid of endogenous Sp1. In addition, other transcription factor sites were unable to substitute for Sp1 in sterol regulation when placed next to the SREBP-binding site. These studies together with recent data from others provide the basis of a working model for sterol regulation of the LDL receptor promoter. The presence of Sp1 sites in several other regulated promoters suggests that this universal transcription factor has been recruited to participate in many regulatory responses possibly by a similar mechanism.

A critical role for cAMP response element-binding protein (CREB) as a Co-activator in sterol-regulated transcription of 3-hydroxy-3-methylglutaryl coenzyme A synthase promoter.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, a key regulatory enzyme in the pathway for endogenous cholesterol synthesis, is a target for negative feedback regulation by cholesterol. When cellular sterol levels are low, the sterol regulatory element-binding proteins (SREBPs) are released from the endoplasmic reticulum membrane, allowing them to translocate to the nucleus and activate SREBP target genes. However, in all SREBP-regulated promoters studied to date, additional co-regulatory transcription factors are required for sterol-regulated activation of transcription. We have previously shown that, in addition to SREBPs, NF-Y/CBF is required for sterol-regulated transcription of HMG-CoA synthase. This heterotrimeric transcription factor has recently been shown to function as a co-regulator in several other SREBP-regulated promoters, as well. In addition to cis-acting sites for both SREBP and NF-Y/CBF, the sterol regulatory region of the synthase promoter also contains a consensus cAMP response element (CRE), an element that binds members of the CREB/ATF family of transcription factors. Here, we show that this consensus CRE is essential for sterol-regulated transcription of the synthase promoter. Using in vitro binding assays, we also demonstrate that CREB binds to this CRE, and mutations within the CRE that result in a loss of CREB binding also result in a loss of sterol-regulated transcription. We further show that efficient activation of the synthase promoter in Drosophila SL2 cells requires the simultaneous expression of all three factors: SREBPs, NF-Y/CBF, and CREB. To date this is the first promoter shown to require CREB for efficient sterol-regulated transcription, and to require two different co-regulatory factors in addition to SREBPs for maximal activation.

5' end of HMG CoA reductase gene contains sequences responsible for cholesterol-mediated inhibition of transcription.

Cholesterol homeostasis is maintained by feedback inhibition of transcription of the gene encoding HMG CoA reductase. To study this mechanism, we joined the 5' end of the hamster reductase gene to the coding region for chloramphenicol acetyltransferase (CAT). The chimeric gene produced high levels of CAT activity in mouse L cells; sterols suppressed expression by 70% to 90%. Sequences responsible for both promotion and inhibition of transcription were distributed over 500 bp extending 300 bp upstream of the reductase transcription initiation sites. Any sizable deletion within this region decreased CAT expression in vivo and CAT mRNA transcription in vitro. This region contains five hexanucleotide repeats (CCGCCC or GGGCGG) that occur in promoters of viral and cellular housekeeping genes. Every reductase-CAT plasmid that showed transcriptional activity also showed inhibition by sterols, indicating that the sites for promotion and inhibition of transcription are closely associated.

The TATA homology and the mRNA 5' untranslated sequence are not required for expression of essential adenovirus E1A functions.

Adenovirus E1A encodes a protein that facilitates the transcription of other early viral transcriptional units and is required for virus-induced transformation. To study the function of non-protein-coding DNA sequence at the 5' end of this transcriptional unit, we constructed mutant viruses with deletions in this region. Deletion of sequence just upstream from the TATA homology does not affect the level or sequence of E1A mRNAs. Deletion of the TATA homology decreases the level of E1A mRNAs by a factor of 5-10 and shifts the mRNA 5' ends from the major 5' end found in wild-type transcripts to a set of minor ends found in wild-type E1A mRNAs. This suggests that the TATA homology is required for an efficient transcription initiation mechanism, and that in its absence a less efficient, less precise mechanism is unmasked. Analysis of mRNAs from other early regions establishes that E2 and E3 regions are most dependent on E1A functions for expression of maximal mRNA levels, E4 is less dependent and E1B is the least dependent. Deletion of the TATA homology, a sequence highly conserved among human adenoviruses, and the entire 5' untranslated sequence of the E1A mRNAs decreases neither the rate of virus replication in a host in which E1A expression is required, nor the efficiency of transformation of rat embryo cells.